Post-translationally modified neurotrophins

ABSTRACT

The present invention describes that neurotrophins undergo post-translational modifications, and that these post-translational modifications mediate the pro-apoptotic and/or pro-neurite activity of neurotrophins. These post-translational modifications notably include nitration and the formation of conformationally-different dimers, as well as of abnormal oligomers, such as tetramers and octamers. The invention further relates to compounds that compete with such modified neurotrophins, as well as to compounds that binds to said modified neurotrophins. The invention thus provides useful agents for the treatment of the conditions or diseases involving a chronic pain and/or neuron loss.

FIELD OF THE INVENTION

The invention relates to the field of neurotrophins, and moreparticularly to post-translationally modified neurotrophins, as well asto their biological, biotechnological, medical, clinical, therapeuticand diagnostic applications.

BACKGROUND OF THE INVENTION

Neurotrophins, and more particularly Nerve Growth Factor (NGF), arecritical for the differentiation and survival of specific neuronalpopulations during development, and modulate neural plasticity in themature nervous system [1, 2]. Paradoxically, NGF has also been describedas inducing apoptosis of neurons during development and eliminatesdamaged neurons and glial cells in pathological conditions [3, 4]. NGFhas been described as a mediator of tissue inflammation and chronic pain[5], and as accumulating in several pathologies undergoingneuroinflammation [6-8].

NGF exerts its actions through two non-related transmembrane receptors,the tyrosine kinase receptor TrkA, and the p75 neurotrophin receptor(p75^(NTR)). TrkA is a tyrosine kinase receptor that activateswell-characterized signalling pathways promoting neuronal survival,differentiation and plasticity [2, 9, 10], whereas p75^(NTR) is a memberof the tumor necrosis factor receptor superfamily that can act as adeath receptor signalling apoptosis [4, 11]. In addition, p75^(NTR) canalso act as a co-receptor for Trk A, B, and C, or interact with otherreceptors (sortilin, Nogo-R) to modulate diverse biological effects,including survival, cytoskeleton rearrangement and axonal elongation [4,12].

Hence, native neurotrophins, such as NGF, have been described as capableof inducing p75^(NTR)-dependent apoptosis.

p75^(NTR) is highly expressed in motor neurons at the embryonic stage,but its expression levels gradually ends after birth [13]. Neither TrkAnor p75^(NTR) are expressed by adult motor neurons, although p75^(NTR)can be re-expressed following axotomy [14-16] and in pathologicalconditions involving motor neuron degeneration, such as amyotrophiclateral sclerosis (ALS) [17, 18]. Furthermore, p75^(NTR) has beenimplicated in motor neuron death induced by axotomy [14, 19, 20].Abnormal expression of p75^(NTR) and NGF may contribute to adult motorneuron death observed in ALS transgenic mice overexpressing mutant Cu—Znsuperoxide dismutase (SOD-1) [18, 21-25].

More recently, pro-neurotrophins have emerged as potent inducers ofp75^(NTR)-dependent apoptosis by signalling through a complex formed byp75^(NTR) and sortilin [14, 15].

For example, WO 2005/014039 expresses and follows this“pro-neurotrophin” hypothesis, without envisioning the possibility ofany post-translational modification of the mature neurotrophin: seee.g., page 46 lines 24-28 of the PCT application as published, whichstates <<because endogenous mature NGF were found in only extremely lowconcentrations in the tissue extracts and nearly failed to meet thedetection limit in culture media, NGF precursors (19-21, 28 and 32 kDa)are the more likely mediators of apoptosis for p75NTR-expressing motorneurons>>.

Hence, before the invention, the main hypothesis was thatpro-neurotrophin, such as pro-NGF, was the endogenous factor leading toneuron apoptosis.

SUMMARY OF THE INVENTION

The inventors have identified, isolated and purified a new species ofgrowth factor that mediates inflammation, chronic pain andneuropathology. The inventors have found that during such conditions ordiseases, neurotrophins, such as NGF, react with endogenous nitratingspecies, such as peroxynitrite, leading to the nitration of saidneurotrophins, and more particularly to the formation of nitratedtyrosine and/or tryptophan residues on said neurotrophins, and that sucha nitrative modification of the neurotrophin molecule inducessignificant conformational changes in the native dimer, which in turnpromotes the formation of high molecular weight abnormal oligomers (suchas tetramers and/or octamers) that are comparable to those found indegenerating tissues.

The inventors have thus identified, isolated and purified modifiedneurotrophins, which have a conformation, a structure and biologicalactivities that are strikingly different from those of native unmodifiedneurotrophins.

Hence, the inventors have found that such a modified neurotrophin is akey agent as an inflammatory mediator, and can play an important role inthe induction of chronic pain and in neuron death in neuropathology.

The invention thus relates to such modified neurotrophins, and moreparticularly to nitrated neurotrophins.

The invention further relates to binders, which are compounds orcompositions that are capable of binding to such modified neurotrophins,and more particularly to specific binders, which are capable of bindingto at least one of such modified neurotrophins, without cross-reactingwith the unmodified native neurotrophin(s).

The invention also relates to the biological, biotechnological, medical,clinical, therapeutic, diagnostic applications of such modifiedneurotrophins, and of such binders.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Ribbon diagram of the NGF structure (PDB code 1BET). The two NGFmonomers (green and blue) interact with each other through a largelyhydrophobic interface [39]. The diagram shows the location of the twotyrosine and three tryptophan residues present in mouse NGF. The Tyr andTrp side chains from one monomer (green) are drawn in a stickrepresentation and labeled (pink). Glu11 (red) from the opposite NGFmonomer is also represented.

FIG. 2. Peroxynitrite treatment enhanced NGF apoptotic activity. (A)Pure motor neuron cultures maintained with GDNF (1 ng/mL) were exposedto increasing concentrations of NGF previously treated withperoxynitrite (1 mM; NGF-ONOO⁻) or decomposed peroxynitrite (NGF-ROA).Dashed lines represent the SD of NONE (trophic factor deprivation). (B)Motor neuron cultures were exposed to NGF (10 ng/mL) previously treatedwith the indicated peroxynitrite concentrations. Dashed lines representthe SD of NONE (trophic factor deprivation). (C) Motor neuron cultureswere treated with increasing concentrations of NGF-ROA or NGF-ONOO⁻ (1mM) in the presence of the nitric oxide donor DETA-NONOate (10 μM, NO).Dashed lines represent the SD of NONE (trophic factor deprivation). (D)Motor neuron cultures were exposed to NGF (100 ng/mL), BSA (100 ng/mL),FGF-1 (10 ng/mL) or FGF-2 (10 ng/mL) previously treated withperoxynitrite (1 mM; black bars, ONOO⁻) or decomposed peroxynitrite (1mM; white bars, ROA). Dashed lines represent SD of GDNF. Motor neuronsurvival was determined 48 h after treatment. Data are expressed aspercentage of GDNF, mean±SD. *Significantly different from GDNF(p<0.05).

FIG. 3. The apoptosis mediated by peroxynitrite-treated NGF requiredp75^(NTR) (A) Blocking antibodies to p75 (a-p75, 1:100, Chemicon#AB1554) and the general caspase inhibitor DEVD-fmk (10 μM) preventedmotor neuron death induced by NGF (100 ng/mL) previously treated withperoxynitrite (1 mM; NGF-ONOO⁻). Antibodies to p75^(NTR) were added onceimmediately after motor neuron plating while DEVD-fmk was added every 24hours. Data are expressed as percentage of GDNF, mean±SD. Dashed linesrepresent the SD of GDNF. *Significantly different from GDNF (p<0.05).(B) Antisense and missense oligonucleotides were added to purified motorneuron cultures at the time of plating. 24 h later, cultures wereexposed to NGF-ONOO⁻ (100 ng/mL) or NGF (100 ng/mL) plus DETA-NONOate(10 μM) (NGF+NO). Antisense oligonucleotides (black bars) completelyblocked the loss of motor neurons induced by both treatments, whereasmissense oligonucleotides (white bars) had no effect on neuronalsurvival. Data are expressed as percentage of the respective GDNF,mean±SD. *Significantly different from the respective GDNF (p<0.05). (C)Motor neuron apoptosis induced by NGF-ONOO⁻ requires the endogenousproduction of peroxynitrite. Motor neuron cultures were treated with 1mM peroxynitrite-treated NGF (100 ng/mL) in the presence L-NAME (1 mM)or MnTBAP (100 μM). Dashed lines represent the SD of GDNF. Data areexpressed as percentage of GDNF, mean±SD. *Significantly different fromGDNF (p<0.05). Motor neuron survival was determined in all cases 48 hafter treatment.

FIG. 4. Peroxynitrite induced NGF oligomerization and nitration. (A)SDS-PAGE showing the formation of high molecular weight species of NGFfollowing treatment with increased concentrations of peroxynitrite(ONOO⁻; 0.25 mM to 1 mM). As a control, NGF was treated with decomposedperoxynitrite (1 mM; ROA). NGF was treated with peroxynitrite at aconcentration of 0.2 mg/mL. 10 μg of protein were applied in each laneand electrophoretic separation was performed in 15% polyacrylamide gelsunder denaturing and reducing conditions. Figure shows a representativegel stained with Coomasie Blue. (B) NGF treated with 1 mM peroxynitrite(NGF-ONOO⁻) or its degradation products (NGF-ROA) was analysed bysize-exclusion chromatography coupled to real-time multi-angle lightscattering (MALS). The absolute molar mass versus time (or volume) ofelution was superimposed with the signals from the 90° LS detector.NGF-ROA (blue) eluted as a single peak with a mass corresponding to thedimer (33.0±1.2 KDa). In contrast, NGF-ONOO⁻ eluted as three peakscorresponding to dimer (33.2±0.6 KDa, 85% of the protein), tetramer(68.5±3.5 KDa, 13%) and octamer (125.0±10.0 KDa, 2%). (C) Western blotshowing increase immunoreactivity for nitrotyrosine. 100 ng of NGFtreated as in (A) was analysed by immunoblotting usinganti-nitrotyrosine (anti-NitroTyr) polyclonal antibodies. Afterstripping the membrane was developed with anti-NGF polyclonalantibodies.

FIG. 5. Reverse-phase HPLC chromatograms of native (A) andperoxynitrite-treated (B) NGF. Native NGF eluted as two peaks at 36.3and 38.1 minutes. Peroxynitrite-treatment (1 mM) led to an incompleteseparation of several products. Nonionized nitrotyrosine absorbs at 360nm. Peroxynitrite-treated NGF had increased absorbance at 360 nm (B)however no absorbance at 360 nm was observed in the native NGFchromatogram (A).

FIG. 6. Mass spectrometry of HPLC collected fractions. (A) Electrospraytime-of-flight mass spectrometry of the eluent from native NGF at 36.3minutes revealed a mass of 13,252 Da, corresponding to Chain A of NGF.Mass signal at 13,078 Da is consistent with the lack of the C-terminalarginine residue. (B) Mass spectrometry of the eluent at 38.6 minutesfrom peroxynitrite-treated NGF showed a 89 Da increase in Chain A mass(from 13,252 to 13,341 Da). The eluent at 39.9 minutes corresponded toChain B and also showed an increase of 90 Da (from 12,357 to 12,449 Da).

FIG. 7. Q-T of mass spectrometry of trypsin-digested HPLC collectedfractions. HPLC purified samples were digested, analysed by Q-T of massspectrometry and subsequent MS/MS ion searches were performed by Mascot.(A) Native untreated NGF eluent at 36.3 minutes, (B) eluent fromperoxynitrite-treated NGF at 38.6 minutes showing modified peptides thatindicate the nitration of Tyr52 and Trp99. Similar modifications wereobserved for the eluent at 39.9 minutes from peroxynitrite-treated NGF.Pyro-glutamic acid (Pyro-Glu) is a common modification of glutamine (Q)at the N-terminus of a peptide.

FIG. 8. Tetranitromethane treatment induced NGF nitration andoligomerization. (A) HPLC chromatogram of tetranitromethane(TNM)-treated NGF. NGF treated with 40-fold excess TNM eluted as twopeaks at 37.9 and 39.9 minutes. Absorbance at 360 nm indicated thepresence of nitrotyrosine in the eluted peaks. (B) Deconvoluted spectraof the eluent at 37.9 minutes showed a mass increase in NGF Chain A of45 Da (to 13,297 Da) and 90 Da (to 13,342 Da) compared to that of nativeNGF (13,252 Da; FIG. 5A). Mass signal at 13,168 Da corresponded todouble nitrated Chain A lacking the C-terminal arginine residue.Deconvoluted spectra of the eluent at 39.9 minutes showed similar massshifts for Chain B. (C) Q-T of mass spectrum of trypsin-digested eluentat 37.9 minutes indicated nitration of Tyr52 and Tyr79. Analysis ofeluent at 39.9 minutes indicated the same modification on Chain B. (D)SDS-PAGE showing the formation of high molecular weight species of NGFfollowing the treatment with TNM (NGF-TNM). 100 ng of protein wereanalysed in each lane and electrophoretic separation was performed in15% polyacrilamide gels under denaturing and reducing conditions. Thefigure shows a representative gel silver stained.

FIG. 9. Tetranitromethane-treated NGF induced p75^(NTR)-dependent motorneuron death. (A) Pure motor neuron cultures maintained with GDNF (1ng/mL) were exposed to increasing concentrations of NGF previouslytreated with vehicle or 40-fold excess TNM (NGF-TNM). Dashed linesrepresent the SD of NONE (trophic factor deprivation). (B) Blockingantibodies to p75^(NTR) (a-p75, 1:100, Chemicon #AB1554) prevented motorneuron death induced by NGF-TNM (100 ng/mL). Antibodies to p75^(NTR)were added with NGF-TNM, 3 hours after motor neuron plating. Dashedlines represent the SD of GDNF. Motor neuron survival was determined 48h after treatment. Data are expressed as percentage of GDNF, mean±SD.*Significantly different from GDNF (p<0.05).

FIG. 10. Urate abolished the effect of peroxynitrite on NGF apoptoticactivity. (A) Urate prevented in a dose dependent manner, tyrosinenitration and oligomerization of NGF. NGF was exposed to decomposedperoxynitrite (1 mM; ROA) or peroxynitrite (1 mM) in the presence ofvehicle or increased concentrations of urate (20 to 1000 μM). Samples(100 ng) were analysed by SDS-15% polyacrylamide gel and Western Blotusing a polyclonal antibody to nitrotyrosine. (B) NGF treated withperoxynitrite in the presence of urate did not affect neuronal survival.Motor neuron cultures were exposed to NGF (100 ng/mL) previously treatedwith peroxynitrite (1 mM) in the presence of vehicle (NGF-ONOO⁻) orurate (200 μM; NGF-ONOO⁻-urate). To eliminate the possibility of adirect effect of unreacted urate on motor neuron survival, theconcentration of urate expected to be present in the culture media afteraddition of NGF-ONOO⁻-urate was added to the cultures exposed toNGF-ONOO⁻. Urate (100 nM) did not prevent motor neuron loss induced byNGF-ONOO⁻ (100 ng/mL). Motor neuron survival was determined 48 h aftertreatment. Dashed lines represent SD of GDNF. Data are expressed aspercentage of GDNF, mean±SD. *Significantly different from GDNF(p<0.05).

FIGS. 11 and 12. NGF amino acid sequences

FIG. 11A: mouse NGF (AAA39818; SEQ ID NO:3)

FIG. 11B: human NGF (CAA37703; SEQ ID NO:4)

FIG. 12A: mature mouse NGF (SEQ ID NO:1)

FIG. 12B: mature human NGF (SEQ ID NO:2)

The Tyr (Y) and Trp (W) residues are shown in bold and underlinedcharacters in FIGS. 12A and 12B:

-   -   in FIG. 12A (mature mouse NGF), are shown the Trp21, Tyr52,        Trp76, Tyr79, Trp99 residues of mature mouse NGF (the residue        numbering being computed by reference to the sequence of the        mature neurotrophin protein);    -   in FIG. 12B (mature human NGF), are shown the Trp21, Tyr52,        Trp76; Tyr79 and Trp99 residues of mature human NGF (the residue        numbering being computed by reference to the sequence of the        mature neurotrophin protein).

FIG. 13. Analysis by western blotting of the conditioned media fromresting or stimulated astrocytes (FGF1, 10 ng/mL; and LPS, 5 microg/mL),using a specific antibody that binds to nitrated NGF, withoutcross-reacting with non-nitrated NGF (lane 1: non-nitrated NGF; lane 2:nitrated NGF; lane 3: conditioned media from resting astrocytes; lane 4:conditioned media from stimulated astrocytes).

FIG. 14. Peroxynitrite increases the neurite outgrowth promotingactivity of NGF.

Dorsal root ganglia explants from E15 rat embryos (that express bothTrkA and p75^(NTR)) were cultured in Neurobasal media in the absence oftrophic factors (NONE) or in the presence of NGF (100 ng/mL), NGFtreated with peroxynitrite (100 ng/mL; nitroNGF-P) or NGF treated withtetranitromethane (nitroNGF-TNM). After 24 hours cultures were fixedwith 4% paraformaldehyde and processed for immunofluorescence againstGAP-43. Note that ganglia treated with nitroNGF exhibit increasedneurite growth compared to those treated with NGF.

DETAILED DESCRIPTION

The invention describes that neurotrophins undergo post-translationalmodifications. To the best of the inventors' knowledge, the invention isthe first description of a post-translational modification of aneurotrophin, and more particularly of NGF.

The inventors demonstrate that these post-translational modificationslead to conformationally different dimers, as well as to abnormaloligomers, such as tetramers and octamers.

Such modified neurotrophins differ from unmodified native (healthy)neurotrophins in structure, conformation, and biological activity.

Such modified neurotrophins have a pro-apoptotic effect on motorneurons, and/or a pro-neurite outgrowth effect on sensory ganglia.Furthermore, these modified neurotrophins can exert these effects atvery low concentrations: the potency of a modified neurotrophin toinduce and/or stimulate such effects is highly increased compared to thepotency that may have (if any) the unmodified neurotrophin, from whichit derives, even when such an unmodified neurotrophin is used in thepresence of added exogenous nitric oxide.

The inventors further demonstrate that under in vivo conditions, thesepost-translational modifications notably result from the nitration ofthe mature neurotrophin by endogenous nitrating species, such asperoxynitrite. Peroxynitrite (ONOO⁻), the reaction product from nitricoxide and superoxide radicals, is formed in vivo mostly in pathologicalconditions associated with increased production of nitric oxide.

To the best of the inventors' knowledge, it is also the first time thatsuch modified neurotrophins are identified, isolated and purified.

In a first aspect, the invention thus relates to these modifiedneurotrophins, and more particularly to these nitrated neurotrophins.

Previous studies, such as Frazier et al. 1973 [52], reported thatnitration of NGF by tetranitromethane (TNM) did not modify NGFbiological activity, at least as assessed by induction of neuriteoutgrowth in sensory ganglia. The invention precisely demonstrates that,on the contrary, nitration of NGF or another neurotrophin, by anitration agent, such as TNM and/or peroxynitrite, deeply modifies itsbiological activity.

Before the invention, it has never been figured out that it is apost-translational modification of neurotrophins, such as NGF (namely,nitration and/or abnormal oligomerization of NGF), that mediates thepro-apoptotic activity exerted by NGF on motor neurons. The prior artteaching only encompassed unmodified NGF and/or its precursor forms,such as pro-NGF; see e.g., Pehar et al., 2004 [25].

The post-translationally modified neurotrophins that have beenidentified by the inventors have a motor neuron apoptotic activityand/or a neurite outgrowth activity at very low concentrations. Thepotency of such a modified neurotrophin to induce and/or stimulate motorneuron apoptosis, and/or neurite outgrowth (e.g., from sensory ganglia),is highly increased compared to the potency that may be exerted (if any)by the unmodified native neurotrophin, from which it derives, even whensaid unmodified native neurotrophin is used in the presence of addedexogenous nitric oxide.

The effect(s) of modified neurotrophins, such as a modified NGF, is(are)detectable at very low concentrations. For example, nitrated NGFsignificantly induces motor neuron apoptosis at concentrations as low as1 ng/mL (cf. FIG. 2A and associated comments).

When used in association with nitric oxide, or a source of nitric oxide,the apoptotic activity of a modified neurotrophin is even furtherincreased. For example, nitrated NGF used together with nitric oxid(NGF-ONOO⁻+NO) significantly induces motor neuron loss at only 1 pg/mL(cf. FIG. 2C and associated comments). Nitration of NGF by peroxynitritein vitro increased the potency of NGF to induce apoptosis of motorneurons by 10,000-fold in the presence of nitric oxide.

Furthermore, the activities exerted by a nitrated neurotrophin on motorneuron apoptosis, and/or on neurite outgrowth from sensory ganglia,appear to be rather specific, as nitrated growth factors other thannitrated neurotrophins, such as nitrated FGF, do not exert any apoptoticeffect on motor neurons (e.g., nitro-FGF-1, nitro-FGF-2; cf. FIG. 2D andassociated comments).

The invention thus relates to modified neurotrophins, which are highlypotent inducers and/or stimulators of motor neuron apoptosis and/or ofneurite outgrowth. These modified neurotrophins have been isolated,identified and characterized by the inventors.

The modified neurotrophins can be characterized by the fact that atleast one residue selected from their Tyr and Trp residues comprises atleast one nitro group.

The modified neurotrophins can alternatively or additionally becharacterized by the fact that they are obtainable by post-translationalnitrative modification of a native neurotrophin, by addition on saidnative neurotrophin of at least one nitro group on at least one residueselected from Tyr and Trp residues, wherein said native neurotrophin isa native pro-neurotrophin or a native mature neurotrophin, preferably anative mature neurotrophin.

By native neurotrophin or native pro-neurotrophin, it is herein intendedthe neurotrophin or pro-neurotrophin corresponding to thenaturally-occurring neurotrophin or pro-neurotrophin that is observed ina healthy mammal, or at the very least in a mammal which does not sufferfrom any abnormal motor neuron apoptosis. A native neurotrophin orpro-neurotrophin thus herein corresponds to the normal “healthy”neurotrophin or pro-neurotrophin. Hence, such a native neurotrophin orpro-neurotrophin is not nitrated.

The term “neurotrophin” is herein meant as the group consisting of NGF(nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-3(neurotrophin-3), NT-4 (neurotrophin-4; which is also referred to asNT-4/5, i.e., neurotrophin-4/5) and NT-6 (neurotrophin-6). In theabsence of any further indication, this term encompasses matureneurotrophin, as well as pro-neurotrophin. In the present invention,preferred neurotrophins are mature neurotrophins.

The invention also relates to the conservative fragments and toconservative variants.

A conservative fragment is a fragment which has retained at least onenitro group on at least one residue, preferably on at least one residueselected from Tyr and Trp residues. It has also retained a capacity ofinducing and/or stimulating motor neuron apoptosis and/or neuriteoutgrowth.

A conservative variant derives from a modified neurotrophin or from aconservative fragment of the invention, by at least one amino acidsubstitution and/or deletion and/or addition, but has retained at leastone nitro group on at least one residue, preferably on at least oneresidue selected from Tyr and Trp residues. Said conservative varianthas also retained a capacity of inducing and/or stimulating motor neuronapoptosis and/or neurite outgrowth.

In the present application, unless otherwise stated, the term “modifiedneurotrophin” or “nitrated neurotrophin” encompasses a modifiedneurotrophin of the invention, as well as any conservative fragmentthereof, as well as any conservative variant of such a modifiedneurotrophin or of such a conservative fragment.

Nitrated neurotrophins of the invention may be obtained by contacting anative neurotrophin or a native pro-neurotrophin with a nitration agent.

A conservative fragment of the invention can be obtained:

-   -   by cleavage of a modified neurotrophin of the invention, or by        contacting fragments of a native neurotrophin or of a native        pro-neurotrophin with a nitration agent,    -   whereby a population of candidate fragments is obtained,    -   and    -   by selection among said population of candidate fragments of a        fragment, which has retained at least one nitro group on at        least one residue, preferably on at least one residue selected        from Tyr and Trp residues, and which has a capacity of inducing        and/or stimulating motor neuron apoptosis and/or neurite        outgrowth.

A conservative variant of the invention can be obtained:

-   -   by substitution and/or deletion and/or addition of at least one        amino acid of a modified neurotrophin of the invention or of a        conservative fragment of the invention, or by contacting a        variant of a native neurotrophin, or of a native        pro-neurotrophin, or of a native neurotrophin fragment, or of a        native pro-neurotrophin fragment, with a nitration agent,    -   whereby a population of candidate variant(s) is obtained, and        -   by selection among this population of candidate variant(s)            of a variant that has retained at least one nitro group on            at least one residue, preferably on at least one residue            selected from Tyr and Trp residues, and that has a capacity            of inducing and/or stimulating motor neuron apoptosis and/or            neurite outgrowth.

Any nitration agent that the skilled person may find appropriate can beused. One or several nitration agent(s) may be used. The nitrationagent(s) being used are agent(s), which comprise(s) at least one nitrogroup, and which are capable of inducing the addition of said at leastone nitro group on a neurotrophin protein, or a fragment or variantthereof, preferably on at least one residue selected from the Tyr andTrp residue(s) of said neurotrophin, or fragment or variant thereof.Preferably, said nitration agent is tetranitromethane and/orperoxynitrite.

The neurotrophin to be nitrated (or the fragment or variant thereof) mayoriginate from any source that the skilled person may find appropriate.It may be a natural peptide, polypeptide or protein, or a fragment of anatural polypeptide or protein, or a recombinant peptide, polypeptide orprotein, or a synthetic peptide or polypeptide. Any method of peptide orpolypeptide synthesis that is known to the skilled person can be used.Examples of synthesis methods, such as the Merrifield solid phasesynthesis, can e.g., be found in <<Solid Phase Peptide Synthesis>> (J.M. Steward & J. D. Young, 1969, Ed. W.H. Freeman Co., San Francisco), orin <<(Peptide synthesis>> (M. Bodansky et al. 1976, John Wiley & Sons,2nd Edition).

Natural sources of neurotrophin notably include astrocytic cells, e.g.,type II astrocytic glial cells.

Neurotrophins are also commercially available, such as murine NGF, whichis available from Harlan (Indianapolis; USA).

Recombinant neurotrophins, or neurotrophin fragments or variants, mayalso be produced by the person of average skill in the art, e.g., byinfecting and/or transfecting and/or transforming appropriate host cells(e.g., fibroblast cells) with a cDNA that encodes said neurotrophin orfragment or variant, under conditions appropriate for the expression ofthe protein or polypeptide or peptide encoded by this cDNA. For example,the production of recombinant human NGF has been described in Johnson etal. 1986 (Cell 47(4):545-554; production from mouse fibroblasts), inAllen et al. 2001 (J. Biochem. Biophys. Methods. 47:239-255; productionfrom baculovirus and insect cell systems), in Rattenholl et al. 2001(Eur. J. Biochem. 268:3296-3303; production from a bacterial system).

Said neurotrophin (or a conservative fragment or variant thereof) iscontacted by said nitration agent under conditions (more particularly,under pH and duration conditions) that are appropriate for saidnitration agent to induce the addition of at least one nitro group on atleast one of the Trp and Tyr residues of said neurotrophin, or fragmentor variant.

The capacity of inducing and/or stimulating motor neuron apoptosisand/or neurite outgrowth can be assessed by any means that are availableto the skilled person. For example, the capacity of inducing and/orstimulating neurite outgrowth can be assessed by exposing sensoryganglia to said nitrated neurotrophin, or fragment or variant thereof,and determining the level of neurite outgrowth, so as to determinewhether said exposure to said nitrated neurotrophin (or fragment orvariant thereof) induces or increases the number of neuritis outgrowingfrom said sensory ganglia. To determine whether the number of neuritesincreases, upon exposure to said nitrated neurotrophin (or fragment orvariant thereof), compared to control conditions, a statisticallysignificant increase of the number of neuritis may be observed.

For example, the capacity of inducing and/or stimulating motor neuronapoptosis can be assessed by exposing motor neurons to said nitratedneurotrophin, or fragment or variant thereof, and counting thenon-apoptotic cells, e.g., the cells that display intact neurites longerthan 4-cell bodies in diameter, and/or the apoptotic cells, so as todetermine whether said exposure to said nitrated neurotrophin (orfragment or variant thereof) induces or increases the number of motorneurons undergoing apoptosis. To determine whether the number ofnon-apoptotic cells decreases and/or whether the number of apoptoticcells increases, upon exposure to said nitrated neurotrophin (orfragment or variant thereof), compared to control conditions, astatistically significant decrease of the number of non-apoptotic cellsand/or a statistically significant increase of the number of apoptoticcells, respectively, may be observed.

For such determinations, appropriate controls that does not comprise theexposure to said nitrated neurotrophin (or fragment or variant thereof)are usually made, such as by placing comparable motor neurons or sensoryganglia in comparable experimental conditions, but without exposure tosaid nitrated neurotrophin (or fragment or variant thereof), or byexposure to a control neurotrophin which has been submitted to adecomposed nitration agent (e.g., decomposed peroxynitrite) and which isadded in reverse order addition. Details of illustrative culture andexperimental conditions can be found described in the examples below(see for example FIG. 2A and comments relating thereto, as well as theparagraph entitled “purified motor neuron cultures”).

The term “statistically significant” or “significantly” is herein usedin its usual meaning in the field of statistics (e.g., t test, z test,chi squared value, or F ratio, etc.), i.e., for comparing a value toanother one, and determining whether these values differ from eachother. The term “statistically significant” or “significantly” henceencompasses the fact that the skilled person may take into account thestandard deviation (if any), which measures the amount of spread of datain a frequency distribution. The desired p value is usually set at analpha level of 5%, or at the more stringent alpha level of 1%.

The monomers of each of the neurotrophins share a number of chemicalcharacteristics, including similar molecular sizes (13.2-15.9 kDa, andexceptionally 21 kDa for NT-6), primary sequence identities thatapproach or exceed 50%, isoelectric points in the range 9-10, and sixconserved half-cystines in the same conserved positions that give riseto three intrachain disulfide bonds. These three disulfide bonds form acharacteristic cystine knot (see FIG. 1). Preferably, said neurotrophinis NGF or BDNF. Most preferably, said neurotrophin is NGF.

Amino acid sequences of murine NGF and human NGF are shown in FIGS. 11A,12A (murine NGF) and 11B and 12B (human NGF).

Preferably, said neurotrophin is a human neurotrophin, most preferably ahuman NGF.

The product of the nitration reaction may comprise unreacted compoundsand/or by-products. If desired or necessary, said post-translationalnitrative modification is followed by separation and/or isolation of thenitrated neurotrophin from unreacted compounds and/or by-products.

The product of the nitration reaction may comprise severalmonomer/oligomer species of the nitrated neurotrophin. If desired ornecessary, said post-translational nitrative modification is followed byseparation and/or isolation of each of these different monomer/oligomerspecies, whereby these species are separated and/or isolated from eachother.

Said separation and/or isolation can e.g., be performed by HLPCchromatography, and/or by mass spectrometry of HPLC eluent fractions(electrospray time-of-flight mass spectrometry), whereby each monomer oroligomer species is obtainable in a pure form.

Methods that may be appropriated to obtain one single monomer/oligomerspecies in a pure form may include, liquid separation using sizeexclusion, ion exchange, immunoaffinity or reverse phase chromatography.Electrophoresis in non-denaturalizing conditions and immunoprecipitationmay also be applied.

Said nitration of neurotrophin can be performed by contacting saidneurotrophin with a nitration agent, such as peroxynitrite and/ortetranitromethane, under conditions enabling the addition of at leastone nitro group, preferably on at least one Tyr or Trp residue.

For example, said nitration can be performed in 50 mM sodium phosphatebuffer, pH 7.4, containing 20 mM sodium bicarbonate.

Said neurotrophin can for example be at a concentration of 0.2 to 1mg/mL.

Said neurotrophin may e.g., be subjected to at least one bolus addition(1 microL) of a peroxynitrite solution at a concentration of 0.25 to 2mg/mL in a 0.01M NaOH solution. Several bolus additions may beperformed, e.g., up to ten bolus addition (1 microL each) of saidperoxynitrite solution.

Successive bolus additions of peroxynitrite cause a dose dependentappearance of oligomers of increasing mer-numbers. It can be observede.g., on SDS-PAGE by a dose dependent appearance of three high molecularweight species (see FIG. 4A for nitrated NGF), or by HPLC size-exclusionchromatography coupled to real-time multi-angle light scattering (MALS)(33.2±0.6 kDa for the nitrated NGF dimer; 68.5±3.5 kDa for the nitratedNGF tetramer; 68.5±3.5 kDa for the nitrated NGF octamer; see FIG. 4B).Surprisingly, the peroxynitrite-treated NGF dimer elutes before thenative NGF dimer (as assessed by HPLC size-exclusion chromatographycoupled to MALS analysis), reflecting the existence of conformationalchanges. This dose-dependent appearance of oligomers of increasingmer-numbers is accompanied by a progressive decrease in stainingintensity of native NGF.

Successive bolus additions of peroxynitrite also induce a dose-dependentnitration of the contacted neutrophin (see FIG. 4C for nitrated NGF).

Native NGF elutes as two peaks by reverse-phase HPLC, at 36.3 and 38.1min (see FIG. 5A), corresponding to NGF chain A and NGF chain B.

Nitrated NGF elutes later, for example at 38.6 min or slightly later,due to the presence of NGF species of increased molecular weights.

Hence, in addition to demonstrating that neurotrophins undergoespost-translational modifications, the inventors demonstrate that thesepost-translation modifications notably result from nitration, and moreparticularly from tyrosine nitration. Nitration was not the solepossibility of chemical modification that could account for suchpost-translational modifications.

Indeed, nitration of tyrosine is not the sole oxidative modification oftyrosine that is known to alter the biological activity of a protein:other oxidative modifications in tyrosine are known, such aschlorination, bromination, and hydroxylation to 3-chloro, 3-bromo- or3-hydroxytyrosine (which are promoted with inflammatory conditions).

Other oxidative processes triggered by reactive nitrogen species such asthiol oxidation, disruption of iron-sulfur clusters and oxidation oftransition metal centers can in many cases be more relevant thannitration in the promotion of cell/dysfunction/death.

Furthermore, the role of peroxynitrite in biological nitration has beenrecently questioned (Pfeiffer et al. 2000 J. Biol. Chem. 275:6346-6352;Thomas et al. 2002 Proc. Natl. Acad. Sci. USA 99:12691-12696).

Moreover, the invention describes not only the nitration of tyrosineresidues, but also the nitration of other residues such as tryptophan(as well as, for some neurotrophins, the oxidation of methionine). Theinvention further describes a conformational change in the neurotrophinmolecule, which leads to conformationally-different nitrated dimers, aswell as to abnormal oligomerization (formation of e.g., tetramer andoctamer species).

Most preferably, an isolated form of modified neurotrophin is in amolecular configuration that does not impede its pro-apoptotic activityand/or its pro-neurite outgrowth activity.

The product obtained after nitration of the neurotrophin (or theneurotrophin fragment or variant) can be isolated from the othercomponents of the reaction mixture, e.g., by reverse-phase HPLCchromatography (see e.g., FIG. 5B for nitrated NGF). The eluent(s) thatis(are) thus obtained may comprise one single nitrated neurotrophinoligomer species, or a mixture of nitrated neurotrophin oligomers, butmay be deprived of any other compound other than nitrated neurotrophin.

If desired, such an eluent, or such eluents, may be further purified,e.g., by HPLC chromatography, to isolate the different oligomer speciesfrom each other, or at least to isolate a certain species mixture fromanother species or species mixture. The eluent(s) can be formulated in aform other than a liquid form, e.g., in a solid form. Techniques thatare appropriate for obtaining a solid state formulation of a productthat is initially available in a liquid state, without loosing thestructure and conformation of the product, are known to the skilledperson. Such a technique may e.g., comprises membrane separation and/orevaporation and/or crystallization and/or freeze concentration.

Preferably, an isolated form of modified neurotrophin does not compriseany non-nitrated pro-neurotrophin and/or any non-nitrated matureneurotrophin.

It preferably does not comprise any un-reacted neurotrophin, such asnon-nitrated NGF dimer, nor any remaining nitration agent.Advantageously, it does not comprise any nitrated and/or non-nitratedpro-neurotrophin (pro-NGF, pro-BDNF, pro-NT-3, pro-NT-4). It does moreparticularly not comprise any nitrated and/or non-nitrated pro-NGF(many, if not all of the commercially-available NGF products arebelieved to be in fact a mixture of mature NGF and of pro-NGF). Mostpreferably, it is in a substantially pure form (as defined below), morepreferably in a pure form, which does not contain any compound otherthan nitrated neurotrophin monomer(s) and/or oligomer(s).

An “isolated” protein is a protein, which is substantially separatedfrom other components, which naturally accompany it, e.g., proteins andflanking genomic sequences from the originating species, and which issubstantially separated from other chemicals or compounds that may bepresent in, or formed during the protein synthesis and/or nitrationreactions. The term “isolated” embraces naturally-occurring proteins orpolypeptides, as well as chemically-synthetised proteins orpolypeptides, and proteins or polypeptides synthetised by heterologoussystems.

“Substantially pure” typically means that the protein is isolated fromother compounds, such as contaminating proteins, nucleic acids, or otherbiological compounds derived from the original source organism, or fromchemicals or compounds that are present in, or formed during, theprotein synthesis and/or the nitration reactions. Purity, or“isolation”, may be assayed by standard methods, typically by weight,and will generally be at least about 70% pure, more generally at leastabout 80% pure, often at least about 85% pure, more often at least about90% pure, preferably at least about 95% pure, more preferably at leastabout 98% pure, and in most preferred embodiments, at least 99% pure.Carriers or excipients will often be added, or the formulation may besterile and/or comprise buffer components.

A substantially pure molecule includes isolated forms of the molecule.An isolated protein will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain minor heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

A modified neurotrophin of the invention can consist of a monomer, or ofan oligomer, such as a dimer, a trimer, a tetramer, a pentamer, ahexamer, a heptamer, an octamer. One of the preferred modifiedneurotrophins of the invention is in the form of a monomer, or of adimer, or of tetramer, or of a hexamer, or of an octamer.

Alternatively, a modified neurotrophin of the invention may consist ofat least two different oligomer species, e.g., at least three differentoligomer species. A modified neurotrophin of the invention may thusconsist of at least two, e.g. at least three, different oligomer speciesselected from the group consisting of dimer, trimer, tetramer, pentamer,hexamer, heptamer, octamer.

One of the preferred modified neurotrophins of the invention consists ofat least two different oligomer species selected from dimer, tetramerand octamer. One of the preferred modified neurotrophins of theinvention may thus consist of two oligomer species, such as dimer andtetramer, or dimer and octamer, or tetramer and octamer. Other preferredneurotrophins of the invention may thus consist of three oligomerspecies, such as dimer, tetramer and octamer. Still other preferredneurotrophins of the invention may thus consist of four differentoligomer species, such as dimer, tetramer, hexamer and octamer.

A modified neurotrophin of the invention can be in a solid form, or in aliquid form.

A neurotrophin, such as NGF, BDNF, NT3, NT-4, has a dimeric structureconsisting of two non-covalently linked monomers. When such a dimericstructure is subjected to nitration, nitration of at least one of themonomer, preferably of the two monomers, take place by addition of atleast one nitro group on at least one residue, preferably selected fromthe Tyr and Trp residues.

Hence, when the post-translationally modified product is in solution,aggregates of modified neurotrophin dimers may form, said aggregatesbeing in dynamic mixture, wherein the different oligomer species mayreach equilibrium between each other, to form a mixture of at least twodifferent oligomer structures selected from dimer(s), tetramer(s),hexamer(s), octamer(s).

Said modified neurotrophin may thus be in the form of an oligomermixture consisting of at least two different oligomer structuresselected from dimer, tetramer, hexamer and octamer structures,preferably from dimer, tetramer, and octamer structures.

Dependent on the particular nitration conditions and/or of theparticular nitration agent being used, the resulting product maynevertheless consist of a single oligomer species. For example, when theconcentration of neurotrophin starting material is low, the resultingmodified neurotrophin product may consist of a single species ofnitrated neurotrophin oligomer, notably of a single species of nitratedneurotrophin dimer. For example, when NGF is used at a concentration of0.2-0.4 mg/mL as starting material, the product resulting from nitration(e.g., by peroxynitrite and/or tetranitromethane) may consist of asingle species of nitrated NGF dimer. Such a modified neurotrophin dimernot only differs from the native (healthy) NGF dimer by the presence ofat least one nitro group, but also by a different molecularconformation. It is evidenced, for example, by HPLC size-exclusionchromatography coupled to real-time multi-angle light scattering (MALS)analysis, notably for the modified NGF dimer (see FIG. 4B). The modifiedneurotrophin dimer further shows a biological activity that isdrastically different from the one of the unmodified native neurotrophindimer from which it derives, as the modified neurotrophin dimer iscapable of inducing and/or stimulating the apoptosis of motor neuronsand/or the outgrowth of neuritis, whereas the unmodified nativeneurotrophin dimer does not show such an activity, or only at a very lowlevel, which is significantly inferior to the one observed with thenitrated neurotrophin dimer. For example, the nitrated NGF dimer of theinvention has an apoptotic activity that is about 10,000 fold higherthan the one of the unmodified native NGF dimer from which it derives.

Said neurotrophin, from which the modified neurotrophin derives, can beBDNF, NT-3, NT-4/5 and/or NT-6. Preferably, it is mature BDNF, matureNT-3, mature NT-4/5 or mature NT-6.

Alternatively, said neurotrophin, from which the modified neurotrophinderives, can be NGF. Preferably, it is mature NGF.

NGF is a homodimer of approximately 27 kDa [38,39]. The mouse NGFmonomer has 118 amino acids, although shorter chains truncated at bothN- and C-termini were also identified [38, 40]. Mouse NGF contains twotyrosine residues at positions 52 and 79 (see FIG. 1) [4]). Conserved inall members of the neurotrophin family, Tyr52 participates inhydrophobic contacts at the dimer interface and is also engaged inp75^(NTR) binding [39, 42]. Site-directed mutagenesis studies revealedthe structural importance of this tyrosine residue in determining astable protein conformation [43]. On the other hand, Tyr79 is conservedin most NGFs, but not in other members of the neurotrophin family [41].In mouse NGF, this tyrosine makes contact with residues of the sameprotomer and could also interact with the N-terminus of the secondprotomer [39]. Since these tyrosine residues are highly conserved,peroxynitrite modification of these residues has important consequencesin NGF biological activity.

Illustrative sequences of a mouse NGF and of a human NGF are shown inFIGS. 11A and 11B, respectively.

The sequences of a mature mouse NGF and of a mature human NGF are shownin FIGS. 12A and 12B, respectively.

In what follows, reference is made to specific NGF residues, moreparticularly to specific Tyr and Trp residues. These residues areidentified by their amino acid position, such as e.g., Tyr52 or Trp99.Residue positions are herein computed by reference to the sequence ofthe mature protein, e.g. for mouse NGF, by reference to the sequence ofSEQ ID NO:1 that is shown in FIG. 12A, or for human NGF, by reference tothe sequence of SEQ ID NO:2 that is shown in FIG. 12B.

FIG. 12A shows the 3 Trp residues and the 2 Tyr residues that arecontained in mouse NGF (Trp21, Tyr52, Trp76, Tyr79, Trp99).

FIG. 12B shows the 3 Trp residues and the 2 Tyr residues that arecontained in human NGF (Trp21, Tyr52, Trp76; Tyr79, Trp99).

Advantageously, a modified neurotrophin of the invention is a nitratedNGF, wherein at least one residue selected from Tyr and Trp residuescomprises at least one nitro group.

Said nitrated NGF can be a nitrated NGF protomer in a pure form, e.g., anitrated NGF chain A, or a nitrated NGF chain B (chain B lacks the eightN terminal residues present in chain A).

Said nitrated NGF can be a nitrated NGF oligomer of nitrated NGF chain Aand/or nitrated NGF chain B, in a pure form.

Such a nitrated NGF oligomer can be a nitrated NGF dimer (consisting oftwo nitrated NGF chains A, or of one nitrated chain A and one nitratedNGF chain B, or of two nitrated chains B). Please note that theperoxynitrite-treated NGF dimer elutes before the native NGF dimer (asassessed by HPLC size-exclusion chromatography coupled to MALSanalysis), reflecting the existence of conformational changes (see FIG.4B).

Such a nitrated NGF oligomer can be a nitrated NGF trimer, a nitratedNGF tetramer, a nitrated NGF pentamer, a nitrated NGF hexamer, anitrated NGF heptamer, a nitrated NGF octamer. It preferably is selectedfrom the group consisting of a nitrated NGF dimer, a nitrated NGFtetramer, a nitrated NGF hexamer, a nitrated NGF octamer. Morepreferably, it is selected from the group consisting of a nitrated NGFdimer, a nitrated NGF tetramer, a nitrated NGF octamer.

As above-mentioned, said nitrated NGF oligomer can be in the form of anoligomer mixture, such as e.g., an oligomer mixture consisting of atleast two different oligomer species selected from dimer, tetramer,hexamer and octamer species, most preferably an oligomer mixtureconsisting of at least two different oligomer species selected fromdimer, tetramer and octamer species, more preferably an oligomer mixtureconsisting of the dimer and the tetramer and the octamer species.

Said at least one residue selected from Tyr and Trp residues thatcomprises at least one nitro group can be a Tyr residue, e.g., Tyr52 orTyr79 of murine NGF, or Tyr52 or Tyr79 of human NGF, or any equivalentresidue from other species. If said neurotrophin is a mouse NGF (SEQ IDNO: 1), said at least one Tyr residue is preferably selected from theTyr52 and the Tyr79 residues of mouse NGF (see FIG. 12A). Morepreferably, said at least one Tyr residue is the Tyr52 residue of mouseNGF.

If said neurotrophin is a human NGF (SEQ ID NO: 2), said at least oneTyr residue is preferably selected from the Tyr52, Tyr79 residues ofhuman NGF (see FIG. 12B). More preferably, said at least one Tyr residueis the Tyr52 residue of human NGF.

Said at least one residue selected from Tyr and Trp residues thatcomprises at least one nitro group can be a Trp residue, e.g., Trp99 orTrp21 or Trp76 of mouse NGF, or Trp99 or Trp21 or Trp76 of human NGF, orany equivalent residue from other species.

The number of Tyr and Trp residues of said neurotrophin that bear anitro group (at least one nitrogroup on each of said residues) can be ofat least two, at least three, at least four, at least five, at leastsix, at least seven, at least eight, or at least nine.

All the Tyr and Trp residues of said neurotrophin may bear at least onenitrogroup.

If said neurotrophin is a mouse NGF, said at least two Tyr residuespreferably are the Tyr52 and the Tyr79 residues of mouse NGF (see FIG.12A).

If said neurotrophin is a human NGF, said at least two Tyr residuespreferably are the Tyr52 and the Tyr79 residues of human NGF (see FIG.12B).

If said neurotrophin is a mouse NGF, said at least one Trp residue ispreferably selected from the Trp99, Trp21 and Trp76 residues of mouseNGF (see FIG. 12A). More preferably, said at least one Trp residue isthe Trp99 or Trp21 residue of mouse NGF. Most preferably, said at leastone Trp residue is the Trp99 residue of mouse NGF.

If said neurotrophin is a human NGF, said at least one Trp residue ispreferably selected from the Trp21, Trp76, Trp99 residues of human NGF(see FIG. 12B). More preferably, said at least one Trp residue is theTrp21 or Trp99 residue of human NGF. Most preferably, said at least oneTrp residue is the Trp99 residue of human NGF.

If said neurotrophin is a mouse NGF, said at least two Trp residues arepreferably selected from the Trp99, Trp21 and Trp76 residues of mouseNGF (see FIG. 12A). More preferably, said at least two Trp residues arethe Trp99 and the Trp21 residues of mouse NGF.

If said neurotrophin is a human NGF, said at least two Trp residues arepreferably selected from the Trp21, Trp76, Trp99 residues of human NGF(see FIG. 12B). More preferably, said at least two Trp residues are theTrp21 and Trp99 residues of human NGF.

If said neurotrophin is a mouse NGF, said at least three Trp residuespreferably are the Trp99, Trp21 and Trp76 residues of mouse NGF (seeFIG. 12A). If said neurotrophin is human NGF, said at least three Trpresidues preferably are the Trp21, Trp76, Trp99 residues of human NGF(see FIG. 12B).

When the number of Tyr and Trp residues that bear a nitro group is of atleast two or higher, these residues can be at least one Tyr residue andat least one Trp residue (at least one nitrogroup on each of saidresidues).

If said neurotrophin is a mouse NGF, said at least one Trp residue ispreferably selected from the Trp99, Trp21 and Trp76 residues of mouseNGF (see FIG. 12A). More preferably, said at least one Trp residue isthe Trp99 or Trp21 residue of mouse NGF. Most preferably, said at leastone Trp residue is the Trp99 residue of mouse NGF.

If said neurotrophin is a mouse NGF, said at least one Tyr residue ispreferably selected from the Tyr52 and the Tyr79 residues of mouse NGF(see FIG. 12A). More preferably, said at least one Tyr residue is theTyr52 residue of mouse NGF. All combinations of these Tyr and Trpresidues are encompassed by the present application. More preferably,said at least one Trp residue is the Trp99 or the Trp21 residue of mouseNGF, and said at least one Tyr residue is the Tyr52 residue of mouseNGF.

TABLE 1 illustrative combinations of mouse NGF Tyr and Trp residuesMouse NGF Trp21 Trp76 Trp99 Tyr52 x x x Tyr79 x x x

If said neurotrophin is a human NGF, said at least one Trp residue ispreferably selected from the Trp21, Trp76, Trp99 residues of human NGF(see FIG. 12B). More preferably, said at least one Trp residue is theTrp21 or Trp99 residue of human NGF. Most preferably, said at least oneTrp residue is the Trp99 residue of mouse NGF.

If said neurotrophin is a human NGF, said at least one Tyr residue ispreferably selected from the Tyr52, Tyr79 residues of human NGF (seeFIG. 12B). More preferably, said at least one Tyr residue is the Tyr52residue of human NGF. All combinations of these Tyr and Trp residues areencompassed by the present application. More preferably, said at leastone Trp residue is the Trp99 or the Trp21 residue of human NGF, and saidat least one Tyr residue is the Tyr52 residue of human NGF.

TABLE 2 illustrative combinations of human NGF Tyr and Trp residuesHuman NGF Trp21 Trp76 Trp99 Tyr52 X X X Tyr79 X X X

A modified neurotrophin of the invention may comprise at least one Tyrand at least two Trp residues that bear a nitro group (at least onenitrogroup on each of said residues).

If said neurotrophin is a mouse NGF, said at least two Trp residues arepreferably selected from the Trp99, Trp21 and Trp76 residues of mouseNGF (see FIG. 12A). More preferably, said at least two Trp residues arethe Trp99 and Trp21 residues of mouse NGF.

If said neurotrophin is a mouse NGF, said at least one Tyr residue ispreferably selected from the Tyr52 and the Tyr79 residues of mouse NGF(see FIG. 12A). More preferably, said at least one Tyr residue is theTyr52 residue of mouse NGF. All combinations of these Tyr and Trpresidues are encompassed by the present application. More preferably,said at least two Trp residues are the Trp99 and the Trp21 residues ofmouse NGF, and said at least one Tyr residue is the Tyr52 residue ofmouse NGF.

If said neurotrophin is a human NGF, said at least two Trp residues arepreferably selected from the Trp21, Trp76, Trp99 residues of human NGF(see FIG. 12B). More preferably, said at least two Trp residues are theTrp21 and Trp99 residues of human NGF.

If said neurotrophin is a human NGF, said at least one Tyr residue ispreferably selected from the Tyr52, Tyr79 residues of human NGF (seeFIG. 12B). More preferably, said at least one Tyr residue is the Tyr52residue of human NGF. All combinations of these Tyr and Trp residues areencompassed by the present application. More preferably, said at leasttwo Trp residues are the Trp99 and the Trp21 residues of human NGF, andsaid at least one Tyr residue is the Tyr52 residue of human NGF.

A modified neurotrophin of the invention may comprise at least two Tyrand at least two Trp residues of said neurotrophin that bear a nitrogroup (at least one nitrogroup on each of said residues).

A modified neurotrophin of the invention may comprise at least one Tyrand at least three Trp residues of said neurotrophin that bear a nitrogroup (at least one nitrogroup on each of said residues).

Of course, any combination of Tyr residue number and of Trp residuenumber is encompassed by the present invention. For example, mouse NGFhas three Trp residues and two Tyr residues (cf. FIG. 12A); and humanNGF has two Tyr residues and three Trp residues (cf. FIG. 12B).

Said nitrated neurotrophin may comprise at least one Met residue that isoxidised (e.g., Met=O). If said neutrophin is mouse NGF, said at leastone Met residue preferably is the Met9 residue of (mature) mouse NGF(see FIG. 12A; residue M at position 9 in SEQ ID NO:1).

A modified neurotrophin of the invention can be in a non-glycosylatedform, or in a glycosylated form.

The invention also relates to binders, which are compounds orcompositions that bind to at least one modified neurotrophin of theinvention, more particularly to at least one nitrated neurotrophin ofthe invention. Nitrated neurotrophins notably comprise nitrated NGF,nitrated BDNF, nitrated NT-3, nitrated NT-4/5 and nitrated NT-6. Anillustrative binder is an antibody.

The invention more particularly relates to specific binders, whichspecifically bind to such modified neurotrophin(s).

Specific binders of the invention notably include those compounds orcompositions that bind to at least one modified neurotrophin, moreparticularly to at least one nitrated neurotrophin (e.g., nitrated NGF,nitrated BDNF, nitrated NT-3, nitrated NT-4/5, nitrated NT-6), withoutbinding to the unmodified native neurotrophin from which said at leastone modified neurotrophin derives (e.g., non-nitrated native NGF,non-nitrated native BDNF, non-nitrated native NT-3, non-nitrated nativeNT-4/5, non-nitrated native NT-6, respectively).

Such a specific binder may bind to e.g., at least two nitratedneurotrophins of the invention (e.g., nitrated NGF and nitrated BDNF),without binding to the two non-nitrated neurotrophin dimers, from whichsaid at least two nitrated forms derive (e.g., the non-nitrated NGF andBDNF dimers).

Most preferably, a specific binder of the invention does not bind to anynon-nitrated neurotrophin (it does not bind to any non-nitrated NGF, toany non-nitrated BDNG, to any non-nitrated NT-3, to any non-nitratedNT-4/5 and to any non-nitrated NT-6).

Preferably, a specific binder of the invention binds to a nitrated NGFof the invention, without binding to the non-nitrated NGF dimer, fromwhich said nitrated NGF derives. Most preferably, such a “nitro-NGF”specific binder does not bind to any non-nitrated NGF dimer. Mostpreferably, such a “nitro-NGF” specific binder does not bind to anynon-nitrated neurotrophin (it does not bind to any non-nitrated NGF, toany non-nitrated BDNF, to any non-nitrated NT-3, to any non-nitratedNT-4/5 and to any non-nitrated NT-6).

Such a “nitro-NGF” specific binder may nevertheless further bind to atleast one nitrated neurotrophin selected from the nitrated BDNF, thenitrated NT-3, the nitrated NT-4/5 and the nitrated NT-6 of theinvention.

Preferably, a binder or specific binder of the invention does not bindto glucose. It thus does preferably not comprise any functional bindingsite for glucose.

Preferably, a binder or specific binder of the invention modulates, mostpreferably inhibits or blocks, the activity of at least one of themodified neurotrophin(s) to which said binder or specific binder binds.Said binder or specific binder may e.g., inhibit or block thepro-apoptotic effect that is exerted by said at least one modifiedneurotrophin on motor neurons, and/or inhibit or block the neuriteoutgrowth stimulation and/or induction effect exerted by said at leastone modified neurotrophin on sensory ganglia.

Preferably, a binder or specific binder of the invention does not bindto the non-nitrated pro-neurotrophin (i.e., the nativepro-neurotrophin), from which the unmodified native mature neurotrophinderives. More preferably, a binder or specific binder of the inventiondoes not bind to any non-nitrated pro-neurotrophin (i.e., to any nativepro-neurotrophin).

A binder or specific binder of the invention may nevertheless bind tonitrated neurotrophin as well as to nitrated pro-neurotrophin.

Other binders or specific binders of the invention may bind to nitratedneurotrophin, without binding to nitrated pro-neurotrophin.

More preferably, a binder or specific binder of the invention does notbind to p75^(NTR) and/or TrkA.

Said binder may e.g., bind to a nitrated epitope selected from QYFFETK(SEQ ID NO: 7), wherein the Tyr residue (Y) in position 2 of theseepitopes bears at least one nitro group. This Tyr residue corresponds toTyr52 of mouse NGF, or to Tyr52 of human NGF. This Tyr residue is highlyconserved.

Said binder or specific binder of the invention may advantageously be anantibody, or a chemical that mimics the function of such an antibody.

Antibody and Antibody Mimics:

The antibody may be a polyclonal (e.g., a polyclonal serum) or amonoclonal antibody, including but not limited to fully assembledantibody, single chain antibody, Fab fragment, and chimeric antibody,humanized antibody. The antibody of present invention may also be usedin combination with other therapeutic agents such as proteins,antibodies, and/or with targeting molecules to specifically target acertain cell type, and/or to detection label, such as a radio-isotope toeasily detect said antibody.

Means enabling to produce antibodies are known to the person of skilledin the art.

Animals can be immunized with a nitrated neurotrophin (or fragment orvariant thereof), or an antigenic functional derivative thereof,according to a known method.

Appropriate animals notably comprise mammals, more particularlynon-human mammals, such as rabbit.

For example, a mammal is injected intraperitoneally or subcutaneouslywith said nitrated neurotrophin (or fragment or variant thereof), or anantigenic functional derivative thereof.

Said nitrated neurotrophin (or fragment or variant thereof), orantigenic functional derivative thereof, may be diluted with, orsuspended in an appropriate volume of PBS (Phosphate-Buffered Saline),physiological saline or the like.

An appropriate volume of a standard adjuvant can be mixed with theproduct, if necessary or desired. Illustrative standard adjuvantsnotably comprise Freund's (complete or incomplete) adjuvant, mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

It may be useful to conjugate said nitrated neurotrophin (or fragment orvariant thereof) to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin (KLH), serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor, by using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydrid or SOCl₂.

The solution is administered to the animals several times, e.g., every 4to 21 days. In addition, an appropriate carrier can also be used uponimmunization with an immunogen.

Polyclonal antibodies are heterogeneous populations of antibodymolecules, which can be derived from the sera of animals immunized withsaid at least one nitrated neurotrophin (or fragment or variantthereof), or an antigenic functional derivative thereof.

Monoclonal antibodies (mAb), which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture.

These include, but are not limited to the hybridoma technique of Kohlerand Milstein (1975) Nature 256:495-497; and U.S. Pat. No. 4,376,110, thehuman B-cell hybridoma technique (Kosbor et al. (1983) Immunology Today4:72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030, andthe EBV-hybridoma technique (Cole et al. (1985) Monoclonal AntibodiesAnd Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies maybe of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing a mAb of this invention may becultivated in vitro or in vivo. Production of mAb of the inventionnotably comprises the collection of immunocytes, such as splenocytes,from an immunized animal, and the fusion of these immunocytes to afusion partner.

As a partner cell to be fused with the above immunocyte, a mammalianmyeloma cell can be used. Examples of a cell line of a myeloma cell thatis preferably used herein include various known cell lines, such as themurine myeloma cell line SP2/0-Ag14, or a fused mousemyeloma/non-malignant B-lymphocyte cell line, such as the ATCC HB8464cell line.

Cell fusion of the above immunocytes with myeloma cells can be basicallyperformed according to a known method, for example, the method of Kohlerand Milstein et al (Kohler. G. and Milstein, C., Methods Enzymol. (1981)73, 3-46).

More specifically, the above cell fusion is performed in a standardnutrition culture solution in the presence of, for example, acell-fusion accelerator. As a cell-fusion accelerator, for example,polyethylene glycol (PEG), hemagglutinating virus of Japan (HVJ) or thelike is used. If desired, an adjuvant such as dimethylsulfoxide can alsobe used by addition to further enhance fusion efficiency.

Any ratio of immunocytes to myeloma cells may be set for use herein. Forexample, it is preferable that the number of immunocytes be 1 to 10times greater than that of myeloma cells. As a culture solution to beused for the above cell fusion, for example, a RPM11640 culture solutionor a MEM culture solution which is appropriate for the growth of theabove myeloma cell line, or other standard culture solutions that areused for this type of cell culture can be used. Moreover, a serum fluidsuch as fetal calf serum (FCS) can be used in combination therewith.

Cell fusion is performed by mixing sufficiently certain amounts of theabove immunocytes and myeloma cells in the above culture solution,adding a PEG (e.g., with an average molecular weight of approximately1000 to 6000) solution (a general concentration of 30 to 60% (w/v))pre-heated at approximately 37° C., and then mixing the solution, so asto form target fused cells (hybridomas). Subsequently, an appropriateculture solution is added successively, and then a step of removing thesupernatant by centrifugation is repeated, so that reagents for cellfusion or the like that is unfavorable for the growth of the hybridomasis removed.

The thus obtained hybridomas are selected by culturing the hybridomas ina standard selective culture solution such as a HAT culture solution (aculture solution containing hypoxanthine, aminopterin and thymidine).Culture in the above HAT culture solution is continued for a time periodsufficient for the cells (unfused cells) other than the targethybridomas to die (normally, several days to several weeks).Subsequently, a standard limiting dilution method is conducted, so thatscreening for and monocloning of hybridomas that produce a targetantibody are performed.

In addition to a method with which the above hybridomas are obtained byimmunizing non-human animals with antigens, desired human antibodieshaving binding activity to said nitrated neurotrophin (or fragment orvariant thereof) can also be obtained (see Japanese Patent Publication(Kokoku) No. 1-59878 B (1989)), by sensitizing in vitro humanlymphocytes with said nitrated neurotrophin (or fragment or variantthereof), or a functional antigenic derivative thereof, and causing thesensitized lymphocytes to fuse with the human-derived myeloma cellshaving a permanent division potential.

The thus prepared hybridomas producing monoclonal antibodies can bepassage-cultured in a standard culture solution, or can be stored for along period in liquid nitrogen.

One example of a method employed to obtain monoclonal antibodies fromthe hybridomas involves culturing the hybridomas and obtainingmonoclonal antibodies in the culture supernatant according to a standardmethod. Another method involves administering the hybridomas to mammalsthat are compatible with the hybridomas to cause them to proliferate,and obtaining monoclonal antibodies in the ascites. The former method issuitable to obtain antibodies of high purity. On the other hand, thelatter method is suitable for the mass production of antibodies.

A monoclonal antibody that can be used in the present invention can be arecombinant monoclonal antibody that is prepared by cloning the antibodygene from the hybridoma, incorporating the gene into an appropriatevector, introducing the vector into a host, and then causing the host toproduce the recombinant monoclonal antibodies by genetic engineeringtechniques (e.g., see Vandamme, A. M. et al., Eur. J. Biochem. (1990)192, 767-775, 1990).

In addition to the above host cell, a transgenic animal or plant canalso be used to produce a recombinant antibody.

In addition to the above antibody, artificially altered gene recombinantantibodies such as chimeric antibodies or humanized antibodies can beused for, for example, lowering heteroantigenicity against a human.These altered antibodies can be produced using a known method.

Chimeric antibodies can e.g., be obtained by ligating the DNA encodingthe antibody V-region to a DNA encoding a human antibody C-region,incorporating the product into an expression vector, and thenintroducing the vector into a host to cause the host to produce theantibodies. Using this known method, chimeric antibodies useful in thepresent invention can be obtained.

Humanized antibodies are also referred to as reshaped human antibodies,which are prepared by grafting an antibody CDR (complementaritydetermining region) of a mammal other than a human, such as a mouse, tothe CDR of a human antibody. The general gene recombination techniquethereof is also known (see European Patent Application Publication EP125023 and WO 96/02576, or any one of their US counterparts, such ase.g., U.S. Pat. No. 6,068,040).

An antibody used in the present invention is not limited to the wholemolecule, and may be a fragment of the antibody or the modified productthereof, as long as it still binds to at least one nitrated neurotrophin(or fragment or variant thereof) and has retained the capacity ofinhibiting and/or blocking the apoptotic effect exerted by said at leastone nitrated neurotrophin (or fragment or variant thereof) on motorneurons, and/or the capacity of inhibiting and/or blocking thestimulation and/or induction effect exerted by said at least onenitrated neurotrophin (or fragment or variant thereof) on sensoryganglia.

Multivalent, preferably bivalent, antibody and a monovalent antibody areincluded. Examples of the fragment of an antibody include Fab, F(ab′)2,Fv, Fab/c having one Fab and a complete Fc, and a single chain Fv (scFv)wherein the Fv of the H-chain or the L-chain is ligated with anappropriate linker. Specifically, an antibody fragment is synthesized bytreating the antibody with an enzyme such as papain or pepsin, or genesencoding these antibody fragments are constructed, the genes areintroduced into expression vectors, and the genes are then expressed byappropriate host cells (see e.g., Rousseaux, J. et al., Methods inEnzymology (1989) 121, 663-669, and Bird, R. E. et al., TIBTECH (1991)9, 132-137).

scFv is obtained by linking the H-chain V-region and the L-chainV-region of antibodies. In the scFv, the H-chain V-region and theL-chain V-region are linked via a linker, or preferably a peptide linker(Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,5879-5883). The H-chain V-region and the L-chain V-region in scFv may bederived from any of those described as antibodies in this specification.As a peptide linker to link the V-regions, for example, anysingle-stranded peptide comprising 12 to 19 amino acid residues is used.

A DNA encoding scFv can be obtained as follows. Amplification isperformed by the PCR method using as templates the entire or DNAportions encoding desired amino acid sequences (of a DNA encoding theH-chain or the H-chain V-region of the above antibody, and a DNAencoding the L-chain or the L-chain V-region), and using a primer pairthat specifies both ends. Amplification is then further performed by acombined use of a DNA encoding a peptide linker portion and a primerpair that specifies to cause both ends to ligate respectively to theH-chain and L-chain.

Furthermore, once a DNA encoding scFv is prepared, expression vectorscontaining the DNAs, and hosts transformed with the expression vectors,can be obtained according to the standard method. In addition, by theuse of the host, scFv can be obtained according to the standard method.

These antibody fragments can be produced using hosts by obtaining thegenes thereof in a manner similar to the above method, and then causingthe expression of the genes. The “antibody” in the present inventionalso encompasses these antibody fragments.

While transgenic mammalian cells (e.g., Chinese hamster ovary cells)grown in culture are the industry standard for producing full lengthmAb, mammalian cells may be less suited for the production of antibodyfragments such as Fab or scFv, and prokaryotic expression systems (e.g.,E. coli) or other eukaryotic expression systems, such as yeast or plantcells, may preferably be used

Furthermore, the antibody used in the present invention may be abispecific antibody, which can also be prepared by genetic engineeringtechniques.

The antibodies expressed and produced as described above can be isolatedfrom the cells or host animals, and purified to a uniform level.Isolation and purification of the antibodies to be used in the presentinvention can be performed using affinity columns. An example of acolumn using a protein A column is a Hyper D, POROS, Sepharose F. F.(Pharmacia). Other standard isolation and purification methods that areemployed for proteins may be used, and there is no limitation regardingtheir use. For example, a chromatography column other than the aboveaffinity column, a filter, ultrafiltration, a method of salting out,dialyses and the like may be appropriately selected and combined foruse, so that antibodies can be isolated and purified (Antibodies ALaboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory,1988).

Chemicals that mimic the function(s) of an antibody can be produced.

There are several approaches to the structure and manufacture ofchemicals that mimic the function(s) of an antibody of the invention.

One approach utilizes an alternative protein framework, such ascytochrome b562, or structures comprising ribonucleic acids (RNA)(Hsieh-Wilson et al. 1996, Acc. Chem. Res. 29:164-170).

Unnatural oligomers, such as benzodiazepines, beta-turn mimics, proteaseinhibitors and purine derivatives have also been tested for theirability to function as antibody mimics.

Unnatural biopolymers, such as oligocarbamates, oligoureas andoligosulfones, have been proposed as antibody mimics.

Molecules with some of the recognition properties of antibodies havebeen created by joining various substituents to scaffolds such asxanthese or cubane, or a calixarene unit. These molecules have multiplepeptide loops as the recognition site, but built around the relativelyrigid organic framework formed by the scaffold.

The invention more particularly relates to those antibody mimics thathave a capacity of inhibiting and/or blocking the apoptotic effectexerted by a nitrated neurotrophin (or fragment or variant thereof) onmotor neurons, and/or of inhibiting and/or blocking the stimulationand/or induction effect exerted by a nitrated neurotrophin (or fragmentor variant thereof) on sensory ganglia.

Biological Systems to Screen for Competitors of a Modified Neurotrophinof the Invention:

There are also several approaches to the structure and manufacture ofbiological systems that can be used to screen molecules that competewith a modified neurotrophin of the invention (or of a conservativefragment or variant thereof).

One of the preferred systems is a ribosomal display system. A ribosomaldisplay system comprises at least one ribosome, at least one protein andat least one mRNA encoding this protein. Advantageous ribosomal displaysystems have been described in Hanes and Pluckthun 1997, Proc. Natl.Acad. Sci. USA, vol. 94, pages 4937-4942. Such ribosomal display systemscan be applied to an antibody of the invention, more particularly to aspecific antibody of the invention, advantageously to a scFv of theinvention, more particularly to a specific scFv of the invention.

The invention thus also relates to a ribosomal display system, whichconsists of at least one ribosome, to which at least one scFv of theinvention (more particularly at least one specific scFv of theinvention), as well as at least one mRNA encoding such a scFv, areattached.

Such biological systems, and more particularly such ribosomal displaysystems, can advantageously be used to screen for compounds that bind tosuch a biological system, and preferably for compounds that compete withat least one modified neurotrophin of the invention (or a conservativefragment or a conservative variant) for binding to said biologicalsystems.

Such compounds are thereby identified and isolated.

Preferred compounds are those which bind to a ribosomal display systemof the invention (which preferably comprises at least one ribosome, atleast one specific scFv of the invention, and at least one mRNA encodingthis scFv), without cross-reacting with (i.e., without binding to)another ribosomal display system, wherein said other ribosomal displaysystem also comprises at least one ribosome, at least one antibody(e.g., a scFv), and at least one mRNA encoding this antibody, butwherein said antibody (said scFv) binds to at least one neurotrophin,without binding to any of the modified neurotrophins that could beobtained from this at least one neutrophin.

The invention more particularly relates to those compounds that have acapacity of modulating, preferably of inhibiting and/or blocking, theapoptotic effect exerted by a nitrated neurotrophin (or fragment orvariant thereof) on motor neurons, and/or of modulating, preferably ofinhibiting and/or blocking, the stimulation and/or induction effectexerted by a nitrated neurotrophin (or fragment or variant thereof) onsensory ganglia.

Such compounds may exert their modulation activity, preferably theirinhibitory or blocking activity, by competing with endogenous nitratedneurotrophins for binding to their endogenous target(s), such as e.g.,TrkA, p75^(NTR), or other receptors or co-receptors, such as sortilin.

Therapeutic and Diagnostic Applications:

A modified neurotrophin of the invention (or a conservative fragment orvariant thereof) has the capacity of inducing and/or stimulating theapoptosis of motor neurons, and/or the neurite outgrowth from sensoryganglia.

Non-conservative fragments of a modified neurotrophin can be produced bythe person of average skilled in the art, e.g., by peptide orpolypeptide synthesis. Non-conservative variants of a modifiedneurotrophin, or of a modified neurotrophin fragment, which derive fromsaid modified neurotrophin, or modified neurotrophin fragment, by atleast one amino acid substitution and/or deletion and/or addition, canalso be produced by the person of average skilled in the art, e.g., bypeptide or polypeptide synthesis.

Such non-conservative fragments or variants have lost the capacity ofinducing and/or stimulating motor neuron apoptosis and/or neuriteoutgrowth, or at the very least do not show a detectable capacity forsuch an induction and/or stimulation when injected into a mammal.

Such a non-conservative fragment or variant may be antigenic on and ofits own, or can be associated with at least one other compound (such asan adjuvant, added in association, or by conjugation), to becomeantigenic, or to have an increased antigenicity.

Such a non-conservative fragment or variant may thus be used as an agentfor active immunization. When administered to a mammal, such a humanbeing, in need thereof, it can induce and/or stimulate an immuneresponse, and more particularly an antibody response, whereby antibodiesbinding to at least one modified neurotrophin of the invention (orconservative fragment or variant thereof) are being produced by saidmammal.

Preferred non-conservative fragments or variants are those which caninduce and/or stimulate an antibody response, whereby antibodies, whichbind to at least one modified neurotrophin of the invention (orconservative fragment or variant thereof) but which do not bind to thenon-modified (healthy) native neurotrophin, are being produced by saidmammal.

Such a non-conservative fragment or variant is useful as an antigen toinduce an immune antibody response for the therapy and/or palliationand/or prevention of the apoptosis of motor neurons and/or the outgrowthof neurite. Hence, such a modified neurotrophin of the invention (or aconservative fragment or variant thereof) is useful as an agent for thetreatment and/or palliation and/or prevention of pain, and/or as anagent for the treatment and/or palliation and/or prevention of aneurodegenerative disease or condition.

Compounds, such as those that can be identified and isolated byscreening with a ribosomal display system of the invention, can competewith the activities of a modified neurotrophin of the invention (or aconservative fragment or variant thereof), thereby blocking and/orinhibiting the apoptosis of motor neurons, and/or the neurite outgrowthfrom sensory ganglia.

When administered to a mammal, such a human being, in need thereof, sucha compound is useful to block and/or inhibit the apoptosis of motorneurons, and/or the neurite outgrowth from sensory ganglia.

A binder of the invention, such an antibody of the invention, and moreparticularly a specific antibody of the invention, binds to a modifiedneurotrophin of the invention (or a conservative fragment or variantthereof).

Such a binder is useful as an agent for passive immunization. Whenadministered to a mammal, such a human being, in need thereof, such abinder will bind to said modified neurotrophin of the invention (or aconservative fragment or variant thereof), thereby blocking and/orinhibiting the apoptosis of motor neurons, and/or the neurite outgrowthfrom sensory ganglia.

Such a binder is useful as agent to block and/or inhibit the apoptosisof motor neurons, and/or the neurite outgrowth from sensory ganglia.

Such a binder is useful as an agent for passive immunization therapyand/or palliation and/or prevention of the apoptosis of motor neuronsand/or the outgrowth of neurite. Hence, such a binder is useful as anagent for the treatment and/or palliation and/or prevention of pain,and/or as an agent for the treatment and/or palliation and/or preventionof a neurodegenerative disease or condition.

The invention also relates to any composition, which comprises:

-   -   at least one element selected from the group comprising the        non-conservative fragments and the non-conservative variants of        the invention, or which comprises    -   at least one of said competing compounds, or which comprises    -   at least one element selected from the group comprising the        binders of the invention (more particularly the specific binders        of the invention, still more particularly the antibodies, the        chemical mimics, and the biological mimics of the invention).

Such compositions may further comprise at least one element selectedfrom excipient, diluent, buffer, pharmaceutical vehicule,physiologically acceptable vehicule, adjuvants.

The invention relates to such pharmaceutical compositions, immunogeniccompositions, immunological compositions, drugs, or vaccines.

When used as antigen for active immunization, at least one elementselected from the group comprising the non-conservative fragments andthe non-conservative variants of the invention, or an antigenicallyfunctional equivalent thereof, may be administered to a mammal,preferably a human, via a variety of routes. When used for passiveimmunization, a binder of the invention of the present invention (andmore particularly a specific binder, e.g. a specific mAb), or anantigenically functional equivalent thereof, may be administered to amammal, preferably a human, via a variety of routes.

These routes notably include orally, parenterally, intraperitoneally,intravenously, intraarterially, topically, transdermally, sublingually,intramuscularly, rectally, transbuccally, intranasally, liposomally, viainhalation, vaginally, intraoccularly, via local delivery (for exampleby catheter or stent), subcutaneously, intraadiposally,intraarticularly, or intrathecally. The antibody may also be deliveredto the host locally (e.g., via stents or catheters) and/or in atimed-release manner.

A binder of the invention (e.g., an Ab), and more particularly aspecific binder of the invention (e.g., a specific mAb), is also usefulfor determining the presence, or the excessive presence, of a modifiedneurotrophin of the invention (or a conservative fragment or variantthereof) in an animal, such as a mammal (e.g. a human being), and moreparticularly in a sample collected from such an animal. The excessivepresence of such a modified neurotrophin of the invention (or aconservative fragment or variant thereof) is indicative of the existenceor of a risk to develop a neurodegenerative disease or condition, or apain condition. A binder of the invention (e.g., an Ab), and moreparticularly a specific binder of the invention (e.g., a specific mAb),is thus also useful for the diagnosis of the existence, or the risk todevelop, a condition or disease involving motor neuron apoptosis and/orneurite outgrowth.

Diseases or conditions wherein it is useful to block and/or inhibit theapoptosis of motor neurons notably comprise neurodegenerative conditionsor diseases, ALS (Amyotrophic Lateral Sclerosis), Alzheimer's disease,Huntington disease's, Multiple Sclerosis, any disease or conditioninvolving a memory deficit and/or a concentration disorder, as well asneuroinflammatory conditions or diseases.

Diseases or conditions wherein it is useful to block and/or inhibit theneurite outgrowth from sensory ganglia notably comprise pain state orconditions or feelings, and more particularly:

-   -   neuropathic pain (more particularly, migraine, chronic migraine,        probable analgesic-abuse headache PMH, primary fibromyalgia        syndrome PFMS, nerve-injury induced neuropathic pain, sciatic        nerve lesions, chronic constriction injury),    -   articular pain (more particularly, osteoarthritic conditions,        osteoarthrosis, osteoarthritis),    -   inflammatory pain,    -   cancer pain.

The invention also relates to any composition, which comprises at leastone element selected from the group comprising the modifiedneurotrophins of the invention, the conservative fragments thereof, andthe conservative variants of such a modified neurotrophin or of such aconservative fragment.

Such compositions may further comprise at least one element selectedfrom excipient, diluent, buffer, pharmaceutical vehicule,physiologically acceptable vehicule, adjuvants.

The invention relates to such pharmaceutical compositions, immunogeniccompositions, immunological compositions, drugs, or vaccines.

Such compositions are useful to induce and/or stimulate motor neuronapoptosis and/or neurite outgrowth.

They are thus useful for the prevention and/or treatment and/orpalliation of diseases or conditions where the stimulation and/orinduction of motor neuron apoptosis and/or of neurite outgrowth isdesired, preferably for the prevention and/or treatment and/orpalliation of diseases or conditions where the stimulation and/orinduction of neurite outgrowth is desired, such as diseases, conditionsor trauma that result in neuropathy and nerve injury, including stroke,spinal cord injuries, and neurodegenerative illnesses.

The present application also relates to a method for the treatmentand/or palliation and/or prevention of such diseases or conditions,which comprises administering to a mammal in need thereof an effectiveamount of an antibody, antibody fragment, or scFv,

wherein said antibody, antibody fragment, or scFv binds to at least onenitro-neurotrophin, wherein said at least one nitro-neurotrophincomprises at least one nitro group on at least one residue selected fromits Tyr and Trp residues, without cross-reacting with a non-nitratedneurotrophin.

The present application also relates to a method for determining whetherthere is an abnormal post-translational modification of a neurotrophinstructure, in a sample suspected of containing such a neurotrophinstructure, which comprises determining whether a binder of theinvention, such as an antibody of the invention, more particularly aspecific binder of the invention, such as a specific antibody of theinvention, binds to a target contained in said sample, whereby such abinding is indicative of the presence in said sample of a neurotrophinstructure that has undergone abnormal post-translational modification.

Said abnormal post-translational modification notably comprisesnitration of said neurotrophin structure, as above explained and asbelow illustrated.

The present application also relates to a method and a kit for thediagnosis of a disease or condition involving motor neuron apoptosisand/or neurite outgrowth, such as the diseases and conditions that areabove-listed.

The kit of the invention comprises at least one binder of the invention,such as at least one antibody of the invention, more particularly atleast one specific binder of the invention, such as at least onespecific antibody of the invention.

The diagnosis method of the invention comprises determining whether abinder of the invention, such as an antibody of the invention, moreparticularly a specific binder of the invention, such as a specificantibody of the invention, binds to a target (i.e., a ligand) containedin the mammal that is subjected to said diagnosis, preferably in arepresentative sample collected from such a mammal (i.e., a sample thatcan be suspected of containing neurotrophin structures), whereby such abinding is indicative of the fact that said mammal has said disease orcondition, or is at high risk of developing such a disease or condition.

In the present application the term “diagnosis” thus encompasses thedetermination of an existing disease or condition, as well as theprediction of its development, or at the very least the evaluation ofthe propensity of the subject to develop such a disease or condition.

Method of Screening:

The invention also relates to a method of screening for compounds thatare capable of inhibiting and/or blocking the apoptotic activity that amodified neurotrophin of the invention (or a conservative fragment orvariant thereof) may have on motor neurons, and/or the neurite outgrowtheffect that a modified neurotrophin of the invention (or a conservativefragment or variant thereof) may have on sensory ganglia.

Candidate compounds can be screened for their capacity of binding to atleast one binder of the invention (e.g., at least one antibody of theinvention), more particularly to at least one specific binder of theinvention (e.g., at least one specific antibody of the invention, suchas a specific mAb of the invention), or to at least one ribosomaldisplay system of the invention, whereby such a binding capacity isindicative of a potential to inhibit and/or block said apoptoticactivity and/or said neurite outgrowth effect.

Preferred compounds are those compounds, which do not bind to a mAb thatis specific of unmodified native neurotrophin (i.e., a mAb, which bindsto unmodified native neurotrophin, without binding to the modifiedneurotrophin that derives therefrom).

The term “comprising”, which is synonymous with “including” or“containing”, is open-ended, and does not exclude additional, unrecitedelement(s), ingredient(s) or method step(s), whereas the term“consisting of” is a closed term, which excludes any additional element,step, or ingredient which is not explicitly recited. The term“essentially consisting of” is a partially open term, which does notexclude additional, unrecited element(s), step(s), or ingredient(s), aslong as these additional element(s), step(s) or ingredient(s) do notmaterially affect the basic and novel properties of the invention.

The term “comprising” (or “comprise(s)”) hence includes the term“consisting of” (“consist(s) of”), as well as the term “essentiallyconsisting of” (“essentially consist(s) of”). Accordingly, the term“comprising” (or “comprise(s)”) is, in the present application, meant asmore particularly encompassing the term “consisting of” (“consist(s)of”), and the term “essentially consisting of” (“essentially consist(s)of”).

Each of the relevant disclosures of all references cited herein isspecifically incorporated by reference. The following examples areoffered by way of illustration, and not by way of limitation.

EXAMPLES Abstract

Nerve growth factor (NGF) overexpression and increased production ofperoxynitrite occur in several neurodegenerative diseases. Weinvestigated whether NGF could undergo post-translational oxidative ornitrative modification that would modulate its biological activity.Compared to native NGF, peroxynitrite-treated NGF showed the exceptionalability to induce p75^(NTR)-dependent motor neuron apoptosis atphysiologically relevant concentrations. While native NGF requires anexternal source of nitric oxide (NO) to induce motor neuron death,peroxynitrite-treated NGF induced motor neuron apoptosis in the absenceof exogenous NO. Nevertheless, NO potentiated the apoptotic activity ofperoxynitrite-modified NGF. Blocking antibodies to p75^(NTR) ordownregulation of p75^(NTR) expression by antisense treatment preventedmotor neuron apoptosis induced by peroxynitrite-treated NGF. Weinvestigated what oxidative modifications were inducing this toxic gainof function and found peroxynitrite induced tyrosine nitration in a dosedependent manner. Moreover, peroxynitrite triggered the formation ofstable high molecular weight oligomers of NGF, although no evidence of3,3′-dityrosine cross-linking was observed. Preventing tyrosinenitration by urate abolished the effect of peroxynitrite on NGFapoptotic activity. These results indicate that the oxidation of NGF byperoxynitrite enhances NGF apoptotic activity through p75^(NTR) 10,000fold. To our knowledge, this is the first known post-translationalmodification that transforms a neurotrophin into an apoptotic agent.

Introduction

We investigated whether NGF could undergo post-translational oxidativeor nitrative modification, altering its functional activity. We reportthat oxidation of NGF by peroxynitrite in vitro causes nitration andinduces the formation of high molecular weight oligomers. Moreover,these oxidative modifications confer the exceptional ability to inducep75^(NTR)-dependent motor neuron apoptosis at physiological relevantconcentrations. Our data show that oxidative stress by peroxynitrite cancritically modulate neurotrophin activity.

Materials and Methods

Peroxynitrite treatment. Peroxynitrite is notably available from Upstate(e.g., Upstate USA, Charlottesville, Va. 22903, USA). Peroxynitriteconcentration was determined spectrophotometrically at 302 nm (ε=1,670M⁻¹ cm⁻¹). Diluted stock solutions were freshly prepared in 0.01 M NaOH.The reactions of NGF (Harlan; Indianapolis, USA) with differentconcentrations of peroxynitrite (0.25 mM to 2 mM) was performed at aprotein concentration of 0.2 mg/mL to 1.0 mg/mL obtaining similarresults. The reaction was performed in 50 mM sodium phosphate buffer, pH7.4, containing 20 mM sodium bicarbonate. NGF was subjected to 10 bolusaddition of peroxynitrite (1 μL each) to reach the desired finalconcentration of peroxynitrite. One bolus of peroxynitrite stocksolution was rapidly added on the top of the test tube and mixed byvortexing for 3 sec. The procedure was repeated 10 times. To exclude apotential non-specific effect of peroxynitrite treatment because of pHchanges or contaminants, control experiments were performed usingdiluted NaOH or decomposed peroxynitrite (reverse order addition, ROA).Treatment of bovine serum albumin (Sigma), FGF-1 (Sigma) and FGF-2 (R&DSystems) with peroxynitrite was performed as described above at aconcentration of 0.2 mg/mL.

NGF nitration by tetranitromethane. The reaction of NGF (Harlan) with a40-fold molar excess of tetranitromethane (Sigma) was performed at aprotein concentration of 1 mg/mL in 0.1 M Tris-HCl buffer, pH 8.0 for 40min. The reaction was finished by passing the reaction mixture through aSephadex G-25 column equilibrated with and developed in 0.05 M ammoniumbicarbonate, pH 8.0. Electrophoretic Analysis and Western Blot. SDS-PAGEwas performed in 15% polyacrylamide minigels under reducing conditions.Samples were boiled for 5 min in LaemLi buffer before running. Proteinswere visualized by Coomassie Blue or Silver staining. For Western Blotanalysis proteins were transferred to nitrocellulose membranes(Hybond-ECL, Amersham). Membranes were blocked for 2 h in blockingsolution (5% BSA, 0.1% Tween 20 in Tris-buffered saline (TBS), pH 7.4)followed by an overnight incubation with the primary antibody diluted inblocking solution. After washing with 0.1% Tween in TBS the membrane wasincubated with peroxidase-conjugated goat anti-rabbit antibody (1:4000;Bio-Rad) for 1 h, and then washed and developed using the ECLchemiluminescent detection system (Amersham). Primary antibodies usedwere anti-NGF-β polyclonal antibody (1:3000; Chemicon) and polyclonalantibody to nitrotyrosine (1:3500; Upstate).

Size exclusion chromatography coupled to multi-angle light scattering.Samples of NGF treated with peroxynitrite (1 mM) or decomposedperoxynitrite at a concentration of 0.5 mg/mL were injected onto aBiosuite 125 HPLC size exclusion column (Waters) connected in line witha DAWN-EOS Multi-Angle Light Scattering Detector and an OptilabRexrefractometer (Wyatt Tech. Corp.). The HPLC column was equilibrated withand developed in 50 mM sodium phosphate, 50 mM sodium sulphate buffer,pH 7.2. The light scattering unit was calibrated followingmanufacturer's instructions. A value of 0.185 mL/g was assumed for thedn/dc of the protein. The detector responses were normalized bymeasuring the signal for monomeric bovine serum albumin. The temperatureof the light scattering unit and of the refractometer were maintained at25° C. The column and all external connections were at ambienttemperature (approximately 25° C.). The flow rate was maintained at 0.5mL/minute throughout the experiments.

Mass Spectrometry Studies. Samples of native NGF or NGF treated withtetranitromethane or peroxynitrite (1 mM) at a concentration of 1 mg/mL,were separated by reverse-phase high pressured liquid chromatography(HPLC). The stationary phase was a C₁₈ reverse phase Supelco column (15cm×4.6 mm, 5 μM). The mobile phase was H₂O/CH₃CN and 0.1%trifluoroacetic acid. A linear gradient increasing 5% to 60% organicover 55 minutes was used to achieve seperation. Samples were collectedby a fraction collector, concentrated in vacuo, and resuspended to bedivided for mass spectrometric analysis for total molecular weightchanges or analysis of the tryptic digest. To find total changes inmolecular weight, collected fractions were resuspended in 30%acetonitrile with 0.1% formic and then directly injected into aWaters/micromass LCT Classic electrospray time of flight massspectrometer. The mobile phase was 30% acetonitrile, 0.1% formic acidwith 5 μL/min flow rate. The capillary voltage was 3.018 kV, the sourcetemperature was 80° C., and sample cone voltage was 45 V. Trypticdigests were performed by resuspension in a 0.1% RapiGest™ solution in50 mM ammonium bicarbonate buffer and was carried out according toRapiGest™ SF Powder protocol. Briefly, samples were reduced with DTT andblocked with IAA before incubating with trypsin (1:50, μg trypsin: μgprotein) overnight at 37° C. TFA was added to digested protein samplesto a final concentration of 0.5% and samples were centrifuged at 13000rpm for ten minutes. A NanoAcquity Waters HPLC system was used to injectsamples onto the Waters Q-tof Ultima Global Mass spectrometer. Sampleswere loaded at 2 μL/min onto a Jupiter C₁₈ trap and then washed for sixminutes with water and 0.1% formic acid at 1 μL/min. Samples were elutedusing a gradient (0.26 μL/min) which began at 2% acetonitrile, 0.1%formic acid and increased organic at −2%/min over 45 minutes. Sampleswere separated over Waters BEH C₁₈ material in a New Objectivepico-frit, 10 cm column and then injected using a Waters Nano Lockspraysource with electrospray capillary voltage at 3.5 kV and source voltage,70 kV. Data dependent tandem mass spectrometry was performed withcollision energy that was dependent upon the m/z of the parent ion. TheMS² spectra were searched using Mascot MS/MS ion search engine and thepeptides reported received scores of identity or above.

Purified motor neuron cultures. Media and sera were purchased fromGibco-Invitrogen. Motor neuron cultures were prepared from embryonic day15 (E15) rat spinal cord by a combination of metrizamide gradientcentrifugation and immunopanning with a monoclonal antibody directedagainst rat p75^(NTR) (mouse mAb Ig192) as previously described [44].Motor neurons were plated at a density of 350 cells/cm² on 4 wellmultidishes (Nunclon) precoated with polyornithine-laminin. Cultureswere maintained in Neurobasal™ medium supplemented with 2% horse serum,25 mM L-glutamate, 25 μM 2-mercaptoethanol, 0.5 mM L-glutamine, and 2%B-27 supplement (Gibco-Invitrogen). Motor neuron survival was maintainedby the addition of GDNF (1 ng/mL; Sigma) to the culture media. Motorneuron death induced by trophic factor deprivation (NONE, without GDNF)was determined in all experiments as a control, and never was greaterthan 50%. Treatments with the different reagents were performed 3 hoursafter motor neuron plating. Motor neuron survival was assessed after 48hours by direct counting of all cells displaying intact neurites longerthan 4-cell bodies in diameter, in a prefixed area of the dish.

Antisense treatment. Treatment with antisense oligonucleotides todown-regulate p75^(NTR) expression was performed as described previously[45]. Briefly, HPLC-purified phosphorothioate antisense and missenseoligonucleotides (5 μM; Integrated DNA Technologies) were added to thecell suspension of purified motor neurons and repeatedly pipetted beforeseeding. The oligonucleotides were present the whole time of culture. Todetermine the efficiency of uptake, cultures were incubated withp75^(NTR) antisense oligonucleotides with a 5′ 56-FAM fluorescent label.Cells were transferred to the heated stage (37° C.) of a Zeiss LSM510confocal microscope with constant 5% CO₂. Fluorescence was imaged with a63× oil immersion objective. Uptake efficiency was >96% in allexperiments. Sequences used were: p75^(NTR) antisense,5′-ACCTGCCCTCCTCATTGCA-3′ (SEQ ID NO: 5) and p75^(NTR) missense,5′-CTCCCACTCGTCATTCGAC-3′ (SEQ ID NO: 6) [45]. The antisense sequenceused has been shown to be effective at inhibiting p75^(NTR)-dependentmotor neuron death in vivo [20].

Statistics. Each experiment was repeated at least three times and dataare reported as mean±SD. Comparison of the means was performed byone-way analysis of variance. Pairwise contrast between means utilizedthe Student-Newman-Keuls test and differences were declaredstatistically significant if p<0.05. All statistics computations wereperformed using the SigmaStat Software (Jandel Scientific).

Secretion of nitro-NGF by stimulated astrocytes. Conditioned media(concentrated 40× by ultra-filtration) from resting or stimulatedastrocytes (FGF1, 10 ng/mL; and LPS, 5 microg/mL) were analysed bywestern blotting using a specific anti-nitro-NGF antibody. Standard NGF(50 ng, Harlan) or nitro-NGF (50 ng) were loaded in the first two lanes.The specific anti-nitro-NGF antibody that has been used is a polyclonalantibody, which has been obtained by immunization of rabbits against thenitro-NGF that is obtained by nitration of the commercially-availablemouse NGF by peroxynitrite and by isolation the nitro-NGF species thatare thereby produced. The specific anti-nitro-NGF antibody has beenselected as binding to nitro-NGF without cross-reacting with thenon-nitrated standard NGF.

Peroxynitrite increases the neurite outgrowth promoting activity of NGF.Dorsal root ganglia explants from E15 rat embryos (that express bothTrkA and p75^(NTR)) were cultured in Neurobasal media in the absence oftrophic factors (NONE) or in the presence of NGF (100 ng/mL), NGFtreated with peroxynitrite (100 ng/mL; nitroNGF-P) or NGF treated withtetranitromethane (nitroNGF-TNM). After 24 hours cultures were fixedwith 4% paraformaldehyde and processed for immunofluorescence againstGAP-43. Note that ganglia treated with nitroNGF exhibit increasedneurite growth compared to those treated with NGF.

Results Oxidation by Peroxynitrite Enhances NGF Apoptotic Activity

In cultures maintained with GDNF (1 ng/mL), motor neurons expressingp75^(NTR) are not sensitive to NGF. However, peroxynitrite-treated NGFinduced motor neuron death at concentrations as low as 1 ng/mL (FIG.2A). NGF treated with decomposed peroxynitrite (ROA) did not affectmotor neuron survival (FIG. 2A). Peroxynitrite treatment enhanced NGFapoptotic activity in a dose-dependent manner, reaching a plateau atconcentrations higher than 0.5 mM peroxynitrite (FIG. 2B). As previouslyreported [25], in the presence of a steady state concentration of nitricoxide <50 nM, generated from the nitric oxide donor DETA-NONOate (10μM), NGF-ROA significantly induced motor neuron loss at concentrationshigher than 10 ng/mL (FIG. 2C). Moreover, in the presence of nitricoxide, peroxynitrite-modified NGF showed increased apoptotic activity,inducing a 33% of motor neuron loss at only 1 pg/mL (FIG. 2C). Theaddition of peroxynitrite treated-BSA, -FGF-1 or -FGF-2 to motor neuroncultures did not induce motor neuron death (FIG. 2D), suggesting aspecific effect of peroxynitrite treated NGF.

Motor neuron loss induced by peroxynitrite-treated NGF was blocked bythe general caspase inhibitor DEVD-fmk (FIG. 3A), indicating theactivation of an apoptotic mechanism. We have previously shown that NGFinduces motor neuron apoptosis by signaling through p75^(NTR [)25]. Theapoptosis induced by peroxynitrite-treated NGF was also dependent onp75^(NTR) activation since it was completely prevented by the additionof blocking antibodies to p75^(NTR) (FIG. 3A) or the down-regulation ofp75^(NTR) expression by antisense treatment (FIG. 3B). As a control,antisense treatment also blocked motor neuron apoptosis induced bynative NGF in the presence of nitric oxide (FIG. 3B). Motor neuronapoptosis induced by different apoptotic stimuli, including NGF,requires the endogenous production of peroxynitrite [25, 46-48]. Motorneuron loss induced by peroxynitrite-treated NGF was prevented by thegeneral nitric oxide synthase (NOS) inhibitor, L-NAME (1 mM) or the SODmimetic and peroxynitrite decomposition catalyst, MnTBAP (100 μM) (FIG.3C), further confirming the execution of a similar apoptotic mechanism.

Peroxynitrite Induces NGF Oligomerization and Nitration

We then analyzed the modifications induced by peroxynitrite treatment onNGF. Exposure of NGF to successive bolus additions of peroxynitritecaused a dose dependent appearance of three high-molecular-weightspecies as revealed by SDS-PAGE. Staining intensity of native NGFprogressively diminished with the increase in peroxynitriteconcentration (FIG. 4A). Treatment of NGF with decomposed peroxynitrite(ROA) failed to induce this migration shift. The formation of NGFoligomers was confirmed in solution by HPLC size-exclusionchromatography coupled to real-time multi-angle light scattering (MALS)analysis (FIG. 4B). NGF treated with decomposed peroxynitrite (NGF-ROA)eluted from the size-exclusion column as a single peak with a masscorresponding to the dimer (33.0±1.2 KDa). In contrast,peroxynitrite-treated NGF eluted as three peaks, likely corresponding todimer (33.2±0.6 KDa), tetramer (68.5±3.5 KDa) and octamer (125.0±10.0KDa). Surprisingly, the peroxynitrite-treated NGF dimer eluted beforethe native dimer (NGF-ROA), reflecting the existence of conformationalchanges due to protein nitration.

Peroxynitrite treatment also induced dose-dependent nitration of NGF, asrevealed by reactivity with an anti-nitrotyrosine antibody (FIG. 4C).The specific sites of oxidative modifications were determined by massspectrometry of purified oxidation products. Native NGF eluted as twopeaks by reverse-phase HPLC, at 36.3 and 38.1 minutes (FIG. 5A), bothidentified as the NGF polypeptide chain by mass spectrometry. Chain Blacks the eight N terminals residues present in Chain A and is known tobe formed due to limited proteolysis during NGF purification [40].Oxidation of NGF by peroxynitrite resulted in the incomplete separationof several products as eluted by reverse-phase HPLC (FIG. 5B). Massspectrometry of the peroxynitrite-treated fraction collected at 38.6minutes revealed several species of increased molecular weight ascompared to the mass spectrum of unmodified NGF (FIG. 6). As it has beenpreviously described [40], some of the unmodified NGF chains lacked theC-terminal arginine residue (FIG. 6A). In peroxynitrite-treated NGF, thesmallest mass shift was a ˜90 Da increase of Chain A (from 13,252 Da to13,341 Da), suggesting the addition of two nitro groups (45 Da each)(FIG. 6B). In addition, peroxynitrite treatment appeared to induceseveral additional modifications, suggesting up to five nitro groups andmethionine oxidation. NGF contains three tryptophans and two tyrosinesthat might account for the five sites of nitration (Inserted Table ofFIG. 6B). A similar pattern of modifications was observed for chain B ofNGF.

To identify the specific residue(s) undergoing oxidative modification,HPLC purified samples were digested and analysed by mass spectrometry.The comparison of untreated and peroxynitrite-treated NGF digestsrevealed the nitration of the two tyrosine residues, Tyr52 and Trp99(FIG. 7), which may account for the detected total molecular weightchange of 90 Da. Oxidation of NGF by peroxynitrite also resulted in lossof tryptophan fluorescence, supporting the nitration of tryptophan.Other residues of oxidative modification were not detected from thedigested samples, possibly indicating the ion m/z=13,341 correspondingto NGF with nitrated Tyr52 and Trp99, to be the most abundant species.However this could also be due to an increased efficiency of ionizationas compared to other peptides. Importantly, formation of 3,3′-dityrosineupon NGF oxidation was not observed by mass spectrometry, and theabsence of this adduct was further confirmed by the lack of fluorescenceat excitation/emission 320/410 nm.

Does Tyrosine Nitration Alter the Biological Activity of NGF?

In order to ascertain whether tyrosine nitration was critical formodifying NGF biological activity, we treated NGF with tetranitromethane(TNM; 40-fold excess). TNM is commonly used to form 3-nitrotyrosine atalkaline pH in proteins. In the conditions used, TNM induced onlytyrosine nitration as evidenced by mass spectrometry (see below). NGFtreated with TNM separated into two products eluting at 37.9 and 39.9minutes (FIG. 8A). As revealed by mass spectrometry, TNM treatmentincreased the molecular weight of NGF Chain A by 90 Da (from 13,252 to13,342 Da; FIG. 8B), suggesting that the main product was a doublenitrated species. However, the single nitrated species could also beobserved at 13,297 Da (FIG. 8B). The same pattern was observed for ChainB of NGF. Tandem mass spectrometry of the digested products identifiedTyr52 and Tyr79 as the nitrated residues (FIG. 8C), accounting for thetotal molecular weight change (FIG. 8B). Tryptophan nitration or3,3′-dityrosine formation was not observed in TNM-treated NGF. Theelectrophoretic pattern of NGF treated with 40-fold excess of TNM wascomparable to that observed in peroxynitrite-treated NGF, inducing theformation of NGF oligomers (FIG. 8D).

We then analyzed the effect of TNM-treated NGF on motor neuron survival.Similar to peroxynitrite-treated NGF, TNM-treated NGF induced 32% motorneuron loss in the absence of nitric oxide (FIG. 9A). Motor neuron deathinduced by TNM-treated NGF was also prevented by the addition ofblocking antibodies to p75^(NTR) (FIG. 9B), suggesting the triggering ofthe same apoptotic mechanism. Both nitration and NGF oligomerizationwere found as common modifications between TNM- andperoxynitrite-treated NGF.

To further determine if nitration was conferring to NGF the capabilityof inducing motor neuron death, we treated NGF with peroxynitrite in thepresence of urate. This compound, which is particularly effective atinhibiting nitration by peroxynitrite [49], prevented tyrosine nitrationand oligomerization of NGF in a dose dependent manner (FIG. 10A).Moreover, urate (200 μM) abolished the apoptotic effect ofperoxynitrite-treated NGF (FIG. 10B). As a control, 100 nM urate (theconcentration expected to be present in the culture media after adding100 ng/mL of NGF-ONOO⁻-urate) did not prevent motor neuron loss inducedby peroxynitrite-treated NGF (100 ng/mL) (FIG. 10B), implying thatunreacted urate was not affecting motor neuron survival.

Secretion of nitro-NGF by stimulated astrocytes. The use of a specificantibody that binds to nitrated NGF, without cross-reacting withnon-nitrated NGF, shows immunoreactive bands in the conditioned mediafrom reactive astrocytes (see FIG. 13, which shows that monomeric anddimeric nitrated NGF species in the Western blot analysis of theconditioned media of FGF/LPS-stimulated astrocytes). This analysis showsthat nitro-NGF is secreted under inflammatory conditions.

Neurite outgrowth promoting activity. FIG. 14 illustrates the fact thatnitration of NGF increases the neurite outgrowth promoting activity ofNGF.

Discussion

Because motor neuron death and astrocyte reactivity in ALS have beenassociated with the increased production of reactive oxygen and nitrogenspecies [28, 50, 51], we wished to investigate whether the oxidation ornitration of secreted NGF might enhance its apoptotic activity towardmotor neurons. Oxidation of NGF by peroxynitrite in vitro increased thepotency for inducing apoptosis of motor neurons by 10,000-fold in thepresence of nitric oxide. To the best of our knowledge, this is thefirst report of neurotrophin-elicited cell death in culture atphysiologically relevant concentrations in the pg/mL range. IncreasedNGF levels have been implicated in the progressive death of motorneurons occurring in ALS [23-25]. We previously reported that spinalcord extracts from SOD1^(G93A) ALS mice contain sufficient NGF tostimulate p75^(NTR)-dependent apoptosis of cultured motor neurons in thepresence of an external source of nitric oxide [25]. However, the levelsof NGF measured by ELISA in the degenerating spinal cord fromSOD1^(G93A) mice were in the range of picograms per mL [25], aconcentration 10,000 times lower than necessary for purified NGF toinduce apoptosis in pure motor neuron cultures and similar to thepotency of peroxynitrite-treated NGF.

In a previous study, nitration of NGF by TNM did not modify NGFbiological activity as assessed by induction of neurite outgrowth insensory ganglia [52]. The difference in the expression of NGF receptorsmay account for the apparent contradictory results. Sensory gangliaexpress both TrkA and p75^(NTR [)53, 54] while pure motor neuroncultures express p75^(NTR) without detectable expression of TrkA [55].Nitrated-NGF induced motor neuron apoptosis by a mechanism dependent onp75^(NTR) signalling, as blocking antibodies to p75^(NTR) ordownregulation of p75^(NTR) expression by antisense treatment completelyprevented motor neuron death.

The gain of apoptotic activity by NGF was consistently associated withtyrosine nitration and abnormal oligomerization. Mouse NGF contains onlytwo tyrosine residues at positions 52 and 79, both of which areaccessible to the solvent [41]. The hydroxyl group of Tyr79 is involvedin hydrogen bonding interactions to Glu11 from the opposing NGF monomer(FIG. 1). This interaction will reduce ionization of the tyrosinehydroxyl and thus render the residue less susceptible toradical-mediated oxidation, which may explain why Tyr79 was moreresistant to nitration than Tyr52. The latter was readily nitrated byboth peroxynitrite and TNM. Since Tyr52 is highly conserved andimportant for stabilizing NGF [43], its nitration may induceconformational changes in the protein that could facilitate aberrantprotein interactions leading to oligomerization.

NGF oligomers ranged in size from dimers to octamers as evidenced bysize exclusion chromatography coupled to real time-MALS. When purifiedhigh molecular weight oligomers were subjected to chromatography asecond time, monomeric and dimeric peaks reappeared, indicating theformation of non-covalent oligomers. However, both peroxynitrite and TNMtreatments led to the formation of higher oligomers that were stable inSDS-PAGE gels, implying a non-thiol dependent, covalent cross-linking ofsome subunits. Oligomerization of NGF was effectively prevented byurate. Urate is known to prevent peroxynitrite-induced tyrosinenitration by competing for carbonate and nitrogen dioxide radicals,suggesting radical formation is involved in the formation of oligomers.Although 3,3′-dityrosine cross-linking is one mechanism by whichperoxynitrite can induce protein dimerization [56, 57], larger oligomerswould require at least two different tyrosine residues to becrosslinked. If crosslinking only resulted from the generation oftyrosine radicals, nitration of Tyr52 would inhibit oligomerization.Furthermore, 3,3′-dityrosine could not be detected by fluorescence or bymass spectrometry. Therefore, oligomerization of NGF most likelyinvolved other forms of cross-linking induced by peroxynitrite. In thepresence of carbon dioxide, 30% of peroxynitrite forms carbonate radicalplus nitrogen dioxide, which are both moderately strong oxidants thatreadily oxidize both tyrosine and tryptophan to form radicals [58].Tyrosyl radicals also combine at near diffusion-limited rates withnitrogen dioxide to form 3-nitrotyrosine. Tryptophan oxidation yieldsmultiple products, including N-formyl kynurenine and kynurenine, whichcan crosslink proteins [59]. On the other hand, tyrosyl radicals canalso oxidize other amino acids, including tryptophan and cysteine byintramolecular electron transfer reactions [59, 60]. Within NGF, Tyr52is spatially close to Trp21 (FIG. 1). Therefore, the oxidation of Tyr52might transfer the radical to Trp21 and thereby facilitate covalentcrosslinks between NGF molecules.

Peroxynitrite treated NGF potently stimulated p75-dependent apoptosis inmotor neurons. Tyr52 and Trp21 in NGF are involved in the formation ofthe hydrophobic pocket that docks with the p75^(NTR) cysteine-richdomain 2 (CRD2) [42]. Because Trp21 is a major part of the interfacewith p75^(NTR), its oxidation products in vivo, might form covalentadducts to p75^(NTR) and facilitate aberrant apoptotic signaling.However, other receptors could also be involved in the induction ofapoptosis by peroxynitrite treated NGF. The precursor of NGF (proNGF)binds with lower affinity than NGF to p75^(NTR), but it forms a highaffinity-signalling complex by simultaneously binding to p75^(NTR) andsortilin [61]. Also, p75^(NTR) belongs to the TNFα receptor superfamily[62], and other members of this superfamily are known to requiretrimerization of their intracellular death domains for activation [63,64]. Similarly, NGF oligomers could recruit additional p75 receptors andthereby promote trimerization of its death domain, and thus morestrongly activate apoptotic signalling.

Because peroxynitrite treatment of NGF results in a gain-of-function,only a small fraction of nitrated protein is necessary to elicitapoptotic signalling. Peroxynitrite-treated NGF can be formed inpathological and inflammatory conditions where NGF up-regulationcoincides with increased production of peroxynitrite and other nitratingspecies. The occurrence of oxidatively modified and nitrated NGF in vivooffers an exciting new mechanism by which neurotrophin signalling couldbe subverted under pathological conditions associated with increasedoxidative stress.

List of Abbreviations: ALS, amyotrophic lateral sclerosis; FGF,fibroblast growth factor; GDNF, glial derived neurotrophic factor; HPLC,high pressured liquid chromatography; MALS, multi-angle lightscattering; NGF, nerve growth factor; NO, nitric oxide; NOS, nitricoxide sinthase; ONOO⁻, peroxynitrite; p75^(NTR), p75 neurotrophinreceptor; ROA, reverse order addition; SOD1, superoxide dismutase 1;TNM, tetranitromethane.

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1: An inducer and/or stimulator of motor neuron apoptosis and/or ofneurite outgrowth, which is: a modified neurotrophin in an isolatedform, wherein at least one residue selected from Tyr and Trp residuescomprises at least one nitro group; or a conservative fragment of saidisolated form of modified neurotrophin, wherein said conservativefragment has retained at least one residue selected from Tyr and Trpresidues, and wherein said conservative fragment has retained at leastone nitro group on said at least one residue selected from Tyr and Trpresidues, and has retained a capacity of inducing and/or stimulatingmotor neuron apoptosis and/or neurite outgrowth; or a conservativevariant of said isolated form of modified neurotrophin or of saidconservative fragment, wherein said conservative variant derives fromsaid modified neurotrophin or from said conservative fragment by atleast one amino acid substitution and/or deletion and/or addition, buthas retained at least one residue selected from Tyr and Trp residues,and wherein said conservative variant has retained at least one nitrogroup on said at least one residue selected from Tyr and Trp residues,and has retained a capacity of inducing and/or stimulating motor neuronapoptosis and/or neurite outgrowth. 2: An inducer and/or stimulator ofmotor neuron apoptosis and/or neurite outgrowth, which is: a modifiedneurotrophin, wherein said modified neurotrophin is prepared bypost-translational nitrative modification of a native matureneurotrophin, by addition on said native mature neurotrophin of at leastone nitro group on at least one residue selected from Tyr and Trpresidues, and wherein said modified neurotrophin is in an isolated form;or a conservative fragment of said isolated form of modifiedneurotrophin, wherein said conservative fragment has retained at leastone residue selected from Tyr and Trp residues, and wherein saidconservative fragment has retained at least one nitro group on said atleast one residue selected from Tyr and Trp residues, and has retained acapacity of inducing and/or stimulating motor neuron apoptosis and/orneurite outgrowth; or a conservative variant of said isolated form ofmodified neurotrophin or of said conservative fragment, wherein saidconservative variant derives from said modified neurotrophin or fromsaid conservative fragment by at least one amino acid substitutionand/or deletion and/or addition, but has retained at least one residueselected from Tyr and Trp residues, and wherein said conservativevariant has retained at least one nitro group on said at least oneresidue selected from Tyr and Trp residues, and has retained a capacityof inducing and/or stimulating motor neuron apoptosis and/or neuriteoutgrowth. 3: The inducer and/or stimulator of claim 1, wherein saidmodified neurotrophin is a modified neurotrophin monomer. 4: The inducerand/or stimulator of claim 1, wherein said modified neurotrophin is amodified neurotrophin oligomer. 5: The inducer and/or stimulator ofclaim 4, wherein said modified neurotrophin is a modified neurotrophindimer. 6: The inducer and/or stimulator of claim 4, wherein saidmodified neurotrophin is a modified neurotrophin tetramer, or a modifiedneurotrophin hexamer, or a modified neurotrophin octamer. 7: An inducerand/or stimulator of motor neuron apoptosis and/or neurite outgrowth,which is a mixture of: at least two modified neurotrophin oligomersselected from a modified neurotrophin dimer a modified neurotrophintetramer, a modified neurotrophin hexamer and a modified neurotrophinoctamer, wherein said at least two oligomers are of different oligomerspecies, and/or at least one modified neurotrophin monomer of claim 3,and at least one modified neurotrophin oligomer of selected from amodified neurotrophin dimer a modified neurotrophin tetramer, a modifiedneurotrophin hexamer and a modified neurotrophin octamer. 8: The inducerand/or stimulator of claim 7, which comprises at least one modifiedneurotrophin tetramer, and/or at least one modified neurotrophinhexamer, and/or at least one modified neurotrophin octamer. 9: Theinducer and/or stimulator of claim 1, wherein said isolated form ofmodified neurotrophin does not comprise any non-nitratedpro-neurotrophin. 10: The inducer and/or stimulator of claim 1, whereinsaid isolated form of modified neurotrophin does not comprise anynon-nitrated mature neurotrophin. 11: The inducer and/or stimulator ofclaim 1, which is in a pure form. 12: The inducer and/or stimulator ofclaim 1, which is in a molecular configuration that does not impede itspro-apoptotic activity and/or its pro-neurite outgrowth activity. 13:The inducer and/or stimulator of claim 1, wherein said neurotrophin isNGF, BDNF, NT-3 or NT-4. 14: The inducer and/or stimulator of claim 13,wherein said neurotrophin is mature NGF. 15: The inducer and/orstimulator of claim 13, wherein said neurotrophin is mature BDNF, matureNT-3 or mature NT-4. 16: The inducer and/or stimulator of claim 1,wherein said at least one residue selected from Tyr and Trp residuesthat comprises at least one nitro group is a Tyr residue. 17: Theinducer and/or stimulator of claim 16, wherein said Tyr residue is Tyr52or Tyr79 of murine NGF, or Tyr52 or Tyr79 of human NGF. 18: The inducerand/or stimulator of claim 1, wherein said at least one residue selectedfrom Tyr and Trp residues that comprises at least one nitro group is aTrp residue. 19: The inducer and/or stimulator of claim 18, wherein saidTrp residue is Trp99 or Trp21 or Trp76 of mouse NGF, or is Trp99 orTrp21 or Trp76 of human NGF. 20: The inducer and/or stimulator of claim1, wherein at least two of the Tyr and Trp residues of said neurotrophinbear a nitro group in which at least one nitrogroup is on each of saidresidues. 21: The inducer and/or stimulator of claim 20, wherein said atleast two residues are two Tyr residues, or a Tyr residue and a Trpresidue. 22: The inducer and/or stimulator of claim 20, wherein thenumber of Tyr and Trp residues of said neurotrophin that bear a nitrogroup is of at least three, at least four, at least five, at least six,at least seven, at least eight, or at least nine. 23: The inducer and/orstimulator of claim 1, wherein all of the Tyr and Trp residues of saidneurotrophin bear at least one nitrogroup. 24: The inducer and/orstimulator of claim 1, wherein said modified neurotrophin is in anon-glycosylated form or in a glycosylated form. 25: A non-conservativefragment of an inducer and/or stimulator of claim 1, or anon-conservative variant of an inducer and/or stimulator of claim 1,which derives from said inducer and/or stimulator by at least one aminoacid substitution and/or deletion and/or addition, wherein saidnon-conservative fragment or variant has lost the capacity of inducingand/or stimulating motor neuron apoptosis and/or neurite outgrowth. 26:The non-conservative fragment or variant of claim 25, for use as anantigen to induce an immune antibody response for the therapy and/orpalliation and/or prevention of the apoptosis of motor neurons and/orthe outgrowth of neurite. 27: The non-conservative fragment or variantof claim 25, for use as an agent for the treatment and/or palliationand/or prevention of pain and/or of a neurodegenerative disease orcondition. 28: An antibody that binds to at least one inducer and/orstimulator according to claim 1, without cross-reacting with theunmodified native neurotrophin, from which said inducer and/orstimulator derives. 29: The antibody of claim 28, which is a monoclonalantibody. 30: The antibody of claim 28, which is an antibody that bindsto nitrated NGF. 31: The antibody of claim 28, which is an scFv. 32: Theantibody of claim 28, for use as an agent for passive immunizationtherapy and/or palliation and/or prevention of the apoptosis of motorneurons and/or the outgrowth of neurite. 33: The antibody of claim 28,for use as an agent for the treatment and/or palliation and/orprevention of pain. 34: The antibody of claim 28, for use as an agentfor the treatment and/or palliation and/or prevention of aneurodegenerative disease or condition. 35: A ribosomal display system,which consists of at least one ribosome, to which at least one scFv ofclaim 31, as well as at least one mRNA encoding such a scFv, areattached. 36: A method of screening for compounds that are capable ofinhibiting and/or blocking the apoptotic activity that a inducer and/orstimulator of claim 1 may have on motor neurons, and/or the neuriteoutgrowth effect that a inducer and/or stimulator of claim 1 may have onsensory ganglia, wherein candidate compounds are screened for theircapacity of binding to at least one antibody of that binds to at leastone inducer and/or stimulator according to claim 1, withoutcross-reacting with the unmodified native neurotrophin, from which saidinducer and/or stimulator derives, or to at least one ribosomal displaysystem consisting of at least one ribosome, to which at least one scFv,as well as at least one mRNA encoding such a scFv, are attached, wherebysuch a binding capacity is indicative of a potential to inhibit and/orblock said apoptotic activity and/or said neurite outgrowth effect. 37:A composition which comprises at least one non-conservative fragment ofan inducer and/or stimulator of claim 1 or a non-conservative variant ofan inducer and/or stimulator of claim 1, and/or at least one antibodythat binds to at least one inducer and/or stimulator according to claim1, without cross-reacting with the unmodified native neurotrophin, fromwhich said inducer and/or stimulator derives. 38: The non-conservativefragment or variant according to claim 25, for use in the treatmentand/or prevention and/or palliation of a neurodegenerative condition ordisease, any disease or condition involving a memory deficit and/or aconcentration disorder, as well as neuroinflammatory conditions ordiseases. 39: The non-conservative fragment or variant according toclaim 25, for use in the treatment and/or prevention and/or palliationof: neuropathic pain, articular pain, inflammatory pain, or cancer pain.40: The antibody according to claim 28, for use in the treatment and/orprevention and/or palliation of a neurodegenerative condition ordisease, any disease or condition involving a memory deficit and/or aconcentration disorder, as well as neuroinflammatory conditions ordiseases. 41: The antibody according to claim 28, for use in thetreatment and/or prevention and/or palliation of: neuropathic pain,articular pain, inflammatory pain, or cancer pain. 42: The compositionof claim 37, for use in the treatment and/or prevention and/orpalliation of a neurodegenerative condition or disease, any disease orcondition involving a memory deficit and/or a concentration disorder, aswell as neuroinflammatory conditions or diseases. 43: The composition ofclaim 37, for use in the treatment and/or prevention and/or palliationof: neuropathic pain, articular pain, inflammatory pain, or cancer pain.44: The inducer and/or stimulator of claim 1, for use as an agent usefulfor the induction and/or the stimulation of motor neuron apoptosisand/or for the induction and/or the stimulation of neurite outgrowth.45: The inducer and/or stimulator of claim 1, for use in the treatmentand/or prevention and/or palliation of a disease, condition or traumaselected from the group comprising neuropathy, nerve injury, stroke,spinal cord injury, and neurodegenerative illness. 46: A composition,which comprises at least one inducer and/or stimulator according toclaim
 1. 47: The composition of claim 46, for use in the treatmentand/or prevention and/or palliation of a disease, condition or traumaselected from the group comprising neuropathy, nerve injury, stroke,spinal cord injury, and neurodegenerative illness. 48: An in vitromethod for the diagnosis of a neurodegenerative condition or disease,and/or of a pain state or condition or feeling, which comprises:contacting a biological sample with at least one antibody according toclaim 28, and determining whether said at least one antibody binds to aligand contained in said biological sample, whereby such a binding isindicative or predictive of said disease, state, condition or feeling.